Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

PubMed

Leone, Piero; Galluccio, Michele; Barbiroli, Alberto; Eberini, Ivano; Tolomeo, Maria; Vrenna, Flavia; Gianazza, Elisabetta; Iametti, Stefania; Bonomi, Francesco; Indiveri, Cesare; Barile, Maria

2018-01-06

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L -1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (k cat about 2.8 min -1 ), as well as FAD pyrophosphorolysis in a strictly Mg 2+ -dependent manner. The synthesis of FAD is inhibited by HgCl₂. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1

PubMed Central

Lattka, E.; Eggers, S.; Moeller, G.; Heim, K.; Weber, M.; Mehta, D.; Prokisch, H.; Illig, T.; Adamski, J.

2010-01-01

Fatty acid desaturases (FADS) play an important role in the formation of omega-6 and omega-3 highly unsaturated fatty acids (HUFAs). The composition of HUFAs in the human metabolome is important for membrane fluidity and for the modulation of essential physiological functions such as inflammation processes and brain development. Several recent studies reported significant associations of single nucleotide polymorphisms (SNPs) in the human FADS gene cluster with HUFA levels and composition. The presence of the minor allele correlated with a decrease of desaturase reaction products and an accumulation of substrates. We performed functional studies with two of the associated polymorphisms (rs3834458 and rs968567) and showed an influence of polymorphism rs968567 on FADS2 promoter activity by luciferase reporter gene assays. Electrophoretic mobility shift assays proved allele-dependent DNA-binding ability of at least two protein complexes to the region containing SNP rs968567. One of the proteins binding to this region in an allele-specific manner was shown to be the transcription factor ELK1 (a member of ETS domain transcription factor family). These results indicate that rs968567 influences FADS2 transcription and offer first insights into the modulation of complex regulation mechanisms of FADS2 gene transcription by SNPs. PMID:19546342

A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1.

PubMed

Lattka, E; Eggers, S; Moeller, G; Heim, K; Weber, M; Mehta, D; Prokisch, H; Illig, T; Adamski, J

2010-01-01

Fatty acid desaturases (FADS) play an important role in the formation of omega-6 and omega-3 highly unsaturated fatty acids (HUFAs). The composition of HUFAs in the human metabolome is important for membrane fluidity and for the modulation of essential physiological functions such as inflammation processes and brain development. Several recent studies reported significant associations of single nucleotide polymorphisms (SNPs) in the human FADS gene cluster with HUFA levels and composition. The presence of the minor allele correlated with a decrease of desaturase reaction products and an accumulation of substrates. We performed functional studies with two of the associated polymorphisms (rs3834458 and rs968567) and showed an influence of polymorphism rs968567 on FADS2 promoter activity by luciferase reporter gene assays. Electrophoretic mobility shift assays proved allele-dependent DNA-binding ability of at least two protein complexes to the region containing SNP rs968567. One of the proteins binding to this region in an allele-specific manner was shown to be the transcription factor ELK1 (a member of ETS domain transcription factor family). These results indicate that rs968567 influences FADS2 transcription and offer first insights into the modulation of complex regulation mechanisms of FADS2 gene transcription by SNPs.

Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle

2010-07-23

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. Atmore » the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.« less

Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization

PubMed Central

Gao, Yong-Gui; Suzuki, Hiroaki; Itou, Hiroshi; Zhou, Yong; Tanaka, Yoshikazu; Wachi, Masaaki; Watanabe, Nobuhisa; Tanaka, Isao; Yao, Min

2008-01-01

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed. PMID:18988622

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase*

PubMed Central

Liu, Li-Kai; Abdelwahab, Heba; Martin Del Campo, Julia S.; Mehra-Chaudhary, Ritcha; Sobrado, Pablo; Tanner, John J.

2016-01-01

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2′-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μm. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. PMID:27557658

Loss- and Gain-of-Function Mutations in the F1-HAMP Region of the Escherichia coli Aerotaxis Transducer Aer

PubMed Central

del Carmen Burón-Barral, Maria; Gosink, Khoosheh K.; Parkinson, John S.

2006-01-01

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants. PMID:16672601

Crystal structures and atomic model of NADPH oxidase.

PubMed

Magnani, Francesca; Nenci, Simone; Millana Fananas, Elisa; Ceccon, Marta; Romero, Elvira; Fraaije, Marco W; Mattevi, Andrea

2017-06-27

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex.

PubMed

Toogood, Helen S; van Thiel, Adam; Basran, Jaswir; Sutcliffe, Mike J; Scrutton, Nigel S; Leys, David

2004-07-30

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

Stimulation of Nipah Fusion: Small Intradomain Changes Trigger Extensive Interdomain Rearrangements.

PubMed

Dutta, Priyanka; Siddiqui, Ahnaf; Botlani, Mohsen; Varma, Sameer

2016-10-18

Nipah is an emerging paramyxovirus that is of serious concern to human health. It invades host cells using two of its membrane proteins-G and F. G binds to host ephrins and this stimulates G to activate F. Upon activation, F mediates virus-host membrane fusion. Here we focus on mechanisms that underlie the stimulation of G by ephrins. Experiments show that G interacts with ephrin and F through separate sites located on two different domains, the receptor binding domain (RBD) and the F activation domain (FAD). No models explain this allosteric coupling. In fact, the analogous mechanisms in other paramyxoviruses also remain undetermined. The structural organization of G is such that allosteric coupling must involve at least one of the two interfaces-the RBD-FAD interface and/or the RBD-RBD interface. Here we examine using molecular dynamics the effect of ephrin binding on the RBD-RBD interface. We find that despite inducing small changes in individual RBDs, ephrin reorients the RBD-RBD interface extensively, and in a manner that will enhance solvent exposure of the FAD. While this finding supports a proposed model of G stimulation, we also find from additional simulations that ephrin induces a similar RBD-RBD reorientation in a stimulation-deficient G mutant, V 209 VG → AAA. Together, our simulations suggest that while inter-RBD reorientation may be important, it is not, by itself, a sufficient condition for G stimulation. Additionally, we find that the mutation affects the conformational ensemble of RBD globally, including the RBD-FAD interface, suggesting the latter's role in G stimulation. Because ephrin induces small changes in individual RBDs, a proper analysis of conformational ensembles required that they are compared directly-we employ a method we developed recently, which we now release at SimTK, and show that it also performs excellently for non-Gaussian distributions. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE PAGES

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; ...

2015-09-18

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

PubMed

Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

2014-04-11

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.

Molecular and functional characterization of a fads2 orthologue in the Amazonian teleost, Arapaima gigas.

PubMed

Lopes-Marques, Mónica; Ozório, Rodrigo; Amaral, Ricardo; Tocher, Douglas R; Monroig, Óscar; Castro, L Filipe C

2017-01-01

The Brazilian teleost Arapaima gigas is an iconic species of the Amazon. In recent years a significant effort has been put into the farming of arapaima to mitigate overfishing threats. However, little is known regarding the nutritional requirements of A. gigas in particular those for essential fatty acids including the long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ability to biosynthesize LC-PUFA is dependent upon the gene repertoire of fatty acyl desaturases (Fads) and elongases (Elovl), as well as their fatty acid specificities. In the present study we characterized both molecularly and functionally an orthologue of the desaturase fatty acid desaturase 2 (fads2) from A. gigas. The isolated sequence displayed the typical desaturase features, a cytochrome b 5 -domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. Functional characterization of A. gigas fads2 showed that, similar to other teleosts, the A. gigas fads2 exhibited a predominant Δ6 activity complemented with some capacity for Δ8 desaturation. Given that A. gigas belongs to one of the oldest teleostei lineages, the Osteoglossomorpha, these findings offer a significant insight into the evolution LC-PUFA biosynthesis in teleosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyanagi, Takashi

2005-12-09

NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form canmore » function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.« less

Regulated expression of a repressor protein: FadR activates iclR.

PubMed Central

Gui, L; Sunnarborg, A; LaPorte, D C

1996-01-01

The control of the glyoxylate bypass operon (aceBAK) of Escherichia coli is mediated by two regulatory proteins, IclMR and FadR. IclMR is a repressor protein which has previously been shown to bind to a site which overlaps the aceBAK promoter. FAR is a repressor/activator protein which participates in control of the genes of fatty acid metabolism. A sequence just upstream of the iclR promoter bears a striking resemblance to FadR binding sites found in the fatty acid metabolic genes. The in vitro binding specificity of FadR, determined by oligonucleotide selection, was in good agreement with the sequences of these sites. The ability of FadR to bind to the site associated with iclR was demonstrated by gel shift and DNase I footprint analyses. Disruption of FadR or inactivation of the FadR binding site of iclR decreased the expression of an iclR::lacZ operon fusion, indicating that FadR activates the expression of iclR. It has been reported that disruption of fadR increases the expression of aceBAK. We observed a similar increase when we inactivated the FadR binding site of an iclR+ allele. This result suggests that FadR regulates aceBAK indirectly by altering the expression of IclR. PMID:8755903

Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool

PubMed Central

Zhang, Jian; Frerman, Frank E.; Kim, Jung-Ja P.

2006-01-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrial membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking oxidation of fatty acids and some amino acids to the mitochondrial respiratory chain. Deficiencies in ETF or ETF-QO result in multiple acyl-CoA dehydrogenase deficiency, a human metabolic disease. Crystal structures of ETF-QO with and without bound UQ were determined, and they are essentially identical. The molecule forms a single structural domain. Three functional regions bind FAD, the 4Fe4S cluster, and UQ and are closely packed and share structural elements, resulting in no discrete structural domains. The UQ-binding pocket consists mainly of hydrophobic residues, and UQ binding differs from that of other UQ-binding proteins. ETF-QO is a monotopic integral membrane protein. The putative membrane-binding surface contains an α-helix and a β-hairpin, forming a hydrophobic plateau. The UQ—flavin distance (8.5 Å) is shorter than the UQ—cluster distance (18.8 Å), and the very similar redox potentials of FAD and the cluster strongly suggest that the flavin, not the cluster, transfers electrons to UQ. Two possible electron transfer paths can be envisioned. First, electrons from the ETF flavin semiquinone may enter the ETF-QO flavin one by one, followed by rapid equilibration with the cluster. Alternatively, electrons may enter via the cluster, followed by equilibration between centers. In both cases, when ETF-QO is reduced to a two-electron reduced state (one electron at each redox center), the enzyme is primed to reduce UQ to ubiquinol via FAD. PMID:17050691

Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool.

PubMed

Zhang, Jian; Frerman, Frank E; Kim, Jung-Ja P

2006-10-31

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrial membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking oxidation of fatty acids and some amino acids to the mitochondrial respiratory chain. Deficiencies in ETF or ETF-QO result in multiple acyl-CoA dehydrogenase deficiency, a human metabolic disease. Crystal structures of ETF-QO with and without bound UQ were determined, and they are essentially identical. The molecule forms a single structural domain. Three functional regions bind FAD, the 4Fe4S cluster, and UQ and are closely packed and share structural elements, resulting in no discrete structural domains. The UQ-binding pocket consists mainly of hydrophobic residues, and UQ binding differs from that of other UQ-binding proteins. ETF-QO is a monotopic integral membrane protein. The putative membrane-binding surface contains an alpha-helix and a beta-hairpin, forming a hydrophobic plateau. The UQ-flavin distance (8.5 A) is shorter than the UQ-cluster distance (18.8 A), and the very similar redox potentials of FAD and the cluster strongly suggest that the flavin, not the cluster, transfers electrons to UQ. Two possible electron transfer paths can be envisioned. First, electrons from the ETF flavin semiquinone may enter the ETF-QO flavin one by one, followed by rapid equilibration with the cluster. Alternatively, electrons may enter via the cluster, followed by equilibration between centers. In both cases, when ETF-QO is reduced to a two-electron reduced state (one electron at each redox center), the enzyme is primed to reduce UQ to ubiquinol via FAD.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

PubMed Central

Roberts, David L.; Frerman, Frank E.; Kim, Jung-Ja P.

1996-01-01

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins. PMID:8962055

Dynamics driving function: new insights from electron transferring flavoproteins and partner complexes.

PubMed

Toogood, Helen S; Leys, David; Scrutton, Nigel S

2007-11-01

Electron transferring flavoproteins (ETFs) are soluble heterodimeric FAD-containing proteins that function primarily as soluble electron carriers between various flavoprotein dehydrogenases. ETF is positioned at a key metabolic branch point, responsible for transferring electrons from up to 10 primary dehydrogenases to the membrane-bound respiratory chain. Clinical mutations of ETF result in the often fatal disease glutaric aciduria type II. Structural and biophysical studies of ETF in complex with partner proteins have shown that ETF partitions the functions of partner binding and electron transfer between (a) a 'recognition loop', which acts as a static anchor at the ETF-partner interface, and (b) a highly mobile redox-active FAD domain. Together, this enables the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. This 'conformational sampling' enables ETF to recognize structurally distinct partners, whilst also maintaining a degree of specificity. Complex formation triggers mobility of the FAD domain, an 'induced disorder' mechanism contrasting with the more generally accepted models of protein-protein interaction by induced fit mechanisms. We discuss the implications of the highly dynamic nature of ETFs in biological interprotein electron transfer. ETF complexes point to mechanisms of electron transfer in which 'dynamics drive function', a feature that is probably widespread in biology given the modular assembly and flexible nature of biological electron transfer systems.

Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirkensgaard, Kristine G.; Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark; Hägglund, Per

2009-09-01

The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR,more » HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.« less

Bifunctional Homodimeric Triokinase/FMN Cyclase

PubMed Central

Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

2014-01-01

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

LucY: A Versatile New Fluorescent Reporter Protein

PubMed Central

Auldridge, Michele E.; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, George N.; Mead, David; Steinmetz, Eric J.

2015-01-01

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions. PMID:25906065

LucY: A Versatile New Fluorescent Reporter Protein.

PubMed

Auldridge, Michele E; Cao, Hongnan; Sen, Saurabh; Franz, Laura P; Bingman, Craig A; Yennamalli, Ragothaman M; Phillips, George N; Mead, David; Steinmetz, Eric J

2015-01-01

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.

LucY: A versatile new fluorescent reporter protein

DOE PAGES

Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; ...

2015-04-23

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrastmore » to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.« less

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family.

PubMed

Gerber, J; Mühlenhoff, U; Hofhaus, G; Lill, R; Lisowsky, T

2001-06-29

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

Structures and Mechanism of the Monoamine Oxidase Family

PubMed Central

Gaweska, Helena; Fitzpatrick, Paul F.

2011-01-01

Members of the monoamine oxidase family of flavoproteins catalyze the oxidation of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals. PMID:22022344

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

PubMed Central

Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

2011-01-01

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

Behavioral and Electrophysiological Correlates of Memory Binding Deficits in Patients at Different Risk Levels for Alzheimer's Disease.

PubMed

Pietto, Marcos; Parra, Mario A; Trujillo, Natalia; Flores, Facundo; García, Adolfo M; Bustin, Julian; Richly, Pablo; Manes, Facundo; Lopera, Francisco; Ibáñez, Agustín; Baez, Sandra

2016-06-30

Deficits in visual short-term memory (VSTM) binding have been proposed as an early and specific marker for Alzheimer's disease (AD). However, no studies have explored the neural correlates of this domain in clinical categories involving prodromal stages with different risk levels of conversion to AD. We assessed underlying electrophysiological modulations in patients with mild cognitive impairment (MCI), patients in the MCI stages of familial AD carrying the mutation E280A of the presenilin-1 gene (MCI-FAD), and healthy controls. Moreover, we compared the behavioral performance and neural correlates of both patient groups. Participants completed a change-detection VSTM task assessing recognition of changes between shapes or shape-color bindings, presented in two consecutive arrays (i.e., study and test) while event related potentials (ERPs) were recorded. Changes always occurred in the test array and consisted of new features replacing studied features (shape-only) or features swapping across items (shape-color binding). Both MCI and MCI-FAD patients performed worse than controls in the shape-color binding condition. Early electrophysiological activity (100-250 ms) was significantly reduced in both clinical groups, particularly over fronto-central and parieto-occipital regions. However, shape-color binding performance and their reduced neural correlates were similar between MCI and MCI-FAD. Our results support the validity of the VSTM binding test and their neural correlates in the early detection of AD and highlight the importance of studies comparing samples at different risk for AD conversion. The combined analysis of behavioral and ERP data gleaned with the VSTM binding task can offer a valuable memory biomarker for AD.

Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase.

PubMed

Lee, J; Hofhaus, G; Lisowsky, T

2000-07-14

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin-linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino-terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy-terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy-terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD-linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.

Prediction of FAD binding sites in electron transport proteins according to efficient radial basis function networks and significant amino acid pairs.

PubMed

Le, Nguyen-Quoc-Khanh; Ou, Yu-Yen

2016-07-30

Cellular respiration is a catabolic pathway for producing adenosine triphosphate (ATP) and is the most efficient process through which cells harvest energy from consumed food. When cells undergo cellular respiration, they require a pathway to keep and transfer electrons (i.e., the electron transport chain). Due to oxidation-reduction reactions, the electron transport chain produces a transmembrane proton electrochemical gradient. In case protons flow back through this membrane, this mechanical energy is converted into chemical energy by ATP synthase. The convert process is involved in producing ATP which provides energy in a lot of cellular processes. In the electron transport chain process, flavin adenine dinucleotide (FAD) is one of the most vital molecules for carrying and transferring electrons. Therefore, predicting FAD binding sites in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. We used an independent data set to evaluate the performance of the proposed method, which had an accuracy of 69.84 %. We compared the performance of the proposed method in analyzing two newly discovered electron transport protein sequences with that of the general FAD binding predictor presented by Mishra and Raghava and determined that the accuracy of the proposed method improved by 9-45 % and its Matthew's correlation coefficient was 0.14-0.5. Furthermore, the proposed method enabled reducing the number of false positives significantly and can provide useful information for biologists. We developed a method that is based on PSSM profiles and SAAPs for identifying FAD binding sites in newly discovered electron transport protein sequences. This approach achieved a significant improvement after we added SAAPs to PSSM features to analyze FAD binding proteins in the electron transport chain. The proposed method can serve as an effective tool for predicting FAD binding sites in electron transport proteins and can help biologists understand the functions of the electron transport chain, particularly those of FAD binding sites. We also developed a web server which identifies FAD binding sites in electron transporters available for academics.

Phylogenetic analysis of proteins associated in the four major energy metabolism systems: photosynthesis, aerobic respiration, denitrification, and sulfur respiration.

PubMed

Tomiki, Takeshi; Saitou, Naruya

2004-08-01

The four electron transfer energy metabolism systems, photosynthesis, aerobic respiration, denitrification, and sulfur respiration, are thought to be evolutionarily related because of the similarity of electron transfer patterns and the existence of some homologous proteins. How these systems have evolved is elusive. We therefore conducted a comprehensive homology search using PSI-BLAST, and phylogenetic analyses were conducted for the three homologous groups (groups 1-3) based on multiple alignments of domains defined in the Pfam database. There are five electron transfer types important for catalytic reaction in group 1, and many proteins bind molybdenum. Deletions of two domains led to loss of the function of binding molybdenum and ferredoxin, and these deletions seem to be critical for the electron transfer pattern changes in group 1. Two types of electron transfer were found in group 2, and all its member proteins bind siroheme and ferredoxin. Insertion of the pyridine nucleotide disulfide oxidoreductase domain seemed to be the critical point for the electron transfer pattern change in this group. The proteins belonging to group 3 are all flavin enzymes, and they bind flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Types of electron transfer in this group are divergent, but there are two common characteristics. NAD(P)H works as an electron donor or acceptor, and FAD or FMN transfers electrons from/to NAD(P)H. Electron transfer functions might be added to these common characteristics by the addition of functional domains through the evolution of group 3 proteins. Based on the phylogenetic analyses in this study and previous studies, we inferred the phylogeny of the energy metabolism systems as follows: photosynthesis (and possibly aerobic respiration) and the sulfur/nitrogen assimilation system first diverged, then the sulfur/nitrogen dissimilation system was produced from the latter system.

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Koh; E-mail: idakoh@sci.kitasato-u.ac.jp; Moriguchi, Tomotaka

2005-07-29

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The {alpha} subunit is composed of two domains, contains NAD{sup +}, and binds folinic acid. The {beta} subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10 A apart. The {gamma} subunit is in contact with two domains of {alpha} subunit and has possibly a folate-binding structure. The {delta} subunit contains a single atom of zinc and has a Cys{sub 3}His zinc finger structure. Based on the structures determined and on themore » previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.« less

RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

PubMed Central

Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F.; Potter, Michael F.; Palli, Subba R.

2012-01-01

Background NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. Methodology/Principal Findings The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. Conclusions/Significance These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs. PMID:22347424

RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

PubMed

Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

2012-01-01

NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

An Unusual Role for a Mobile Flavin in StaC-like Indolocarbazole Biosynthetic Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldman, Peter J.; Ryan, Katherine S.; Hamill, Michael J.

2012-10-09

The indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here, we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar K{sub d}, whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar K{sub d} while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD-binding motifs and the complementary StaC-10x. We find that thesemore » mutations mediate both FAD affinity and product specificity, with RebC-10x displaying higher StaC activity than StaC itself. X-ray structures of this StaC catalyst identify the substrate of StaC as 7-carboxy-K252c and suggest a unique mechanism for this FAD-dependent enzyme.« less

Identification of small molecular ligands as potent inhibitors of fatty acid metabolism in Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

Malikanti, Ramesh; Vadija, Rajender; Veeravarapu, Hymavathi; Mustyala, Kiran Kumar; Malkhed, Vasavi; Vuruputuri, Uma

2017-12-01

Tuberculosis (Tb) is one of the major health challenges for the global scientific community. The 3-hydroxy butyryl-CoA dehydrogenase (Fad B2) protein belongs to 3-hydroxyl acetyl-CoA dehydrogenase family, which plays a key role in the fatty acid metabolism and β-oxidation in the cell membrane of Mycobacterium tuberculosis (Mtb). In the present study the Fad B2 protein is targeted for the identification of potential drug candidates for tuberculosis. The 3D model of the target protein Fad B2, was generated using homology modeling approach and was validated. The plausible binding site of the Fad B2 protein was identified from computational binding pocket prediction tools, which ranges from ASN120 to VAL150 amino acid residues. Virtual screening was carried out with the databases, Ligand box UOS and hit definder, at the binding site region. 133 docked complex structures were generated as an output. The identified ligands show good glide scores and glide energies. All the ligand molecules contain benzyl amine pharmacophore in common, which show specific and selective binding interactions with the SER122 and ASN146 residues of the Fad B2 protein. The ADME properties of all the ligand molecules were observed to be within the acceptable range. It is suggested from the result of the present study that the docked molecular structures with a benzyl amine pharmacophore act as potential ligands for Fad B2 protein binding and as leads in Tb drug discovery.

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

PubMed Central

Jiang, Tianyi; Guo, Xiaoting; Yan, Jinxin; Zhang, Yingxin; Wang, Yujiao; Zhang, Manman; Sheng, Binbin; Ma, Cuiqing; Xu, Ping

2017-01-01

ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes. PMID:28847921

Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.

PubMed

Zirak, P; Penzkofer, A; Moldt, J; Pokorny, R; Batschauer, A; Essen, L-O

2009-11-09

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.

Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels

PubMed Central

Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

2011-01-01

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open). PMID:21308847

Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhi; Kim, David D.; Nelson, Ornella D.

2015-10-08

Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation.We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fee4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain andmore » investigated their iron reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fee4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the ironereductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have.« less

Brain Information Sharing During Visual Short-Term Memory Binding Yields a Memory Biomarker for Familial Alzheimer's Disease.

PubMed

Parra, Mario A; Mikulan, Ezequiel; Trujillo, Natalia; Sala, Sergio Della; Lopera, Francisco; Manes, Facundo; Starr, John; Ibanez, Agustin

2017-01-01

Alzheimer's disease (AD) as a disconnection syndrome which disrupts both brain information sharing and memory binding functions. The extent to which these two phenotypic expressions share pathophysiological mechanisms remains unknown. To unveil the electrophysiological correlates of integrative memory impairments in AD towards new memory biomarkers for its prodromal stages. Patients with 100% risk of familial AD (FAD) and healthy controls underwent assessment with the Visual Short-Term Memory binding test (VSTMBT) while we recorded their EEG. We applied a novel brain connectivity method (Weighted Symbolic Mutual Information) to EEG data. Patients showed significant deficits during the VSTMBT. A reduction of brain connectivity was observed during resting as well as during correct VSTM binding, particularly over frontal and posterior regions. An increase of connectivity was found during VSTM binding performance over central regions. While decreased connectivity was found in cases in more advanced stages of FAD, increased brain connectivity appeared in cases in earlier stages. Such altered patterns of task-related connectivity were found in 89% of the assessed patients. VSTM binding in the prodromal stages of FAD are associated to altered patterns of brain connectivity thus confirming the link between integrative memory deficits and impaired brain information sharing in prodromal FAD. While significant loss of brain connectivity seems to be a feature of the advanced stages of FAD increased brain connectivity characterizes its earlier stages. These findings are discussed in the light of recent proposals about the earliest pathophysiological mechanisms of AD and their clinical expression. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mechanistic insights into the inhibition of quercetin on xanthine oxidase.

PubMed

Zhang, Cen; Wang, Rui; Zhang, Guowen; Gong, Deming

2018-06-01

Quercetin, one of the most abundant flavonoid in the daily diet, was found to reversibly inhibit the generation of uric acid and superoxide radicals (O 2 - )catalyzed by xanthine oxidase (XOD) in a mixed-type manner with IC 50 values of (2.74±0.04)×10 -6 and (2.90±0.03)×10 -6 molL -1 , respectively, and the inhibition of quercetin on O 2 - generation may be ascribed to the reduced form of XOD by a ping-pong mechanism. XOD had one high affinity binding site for quercetin with a binding constant of 4.28×10 4 Lmol -1 at 298K, and the binding process was predominately driven by van der Waals forces and hydrogen bonds on account of the negative enthalpy and entropy changes. Moreover, molecular docking confirmed that the binding site for quercetin located in the isoalloxazine ring of the flavin adenine dinucleotide (FAD) domain of XOD, then the diffusion of O 2 - out of the FAD site was blocked in favor of another electron transferred from FADH 2 to O 2 - to form hydrogen peroxide (H 2 O 2 ). This study may clarify the role of quercetin on inhibiting XOD catalysis and provide a potential nutritional supplement for preventing gout and peroxidative damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

PubMed

Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

2017-04-01

The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s -1 . This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

PubMed Central

Sarapusit, Songklod; Lertkiatmongkol, Panida; Duangkaew, Panida; Rongnoparut, Pornpimol

2013-01-01

Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy

PubMed Central

Vonck, Janet; Parcej, David N.; Mills, Deryck J.

2016-01-01

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes. PMID:27458710

Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels.

PubMed

Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

2011-03-01

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open). Copyright © 2011 The Protein Society.

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

PubMed Central

Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

2016-01-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

Discovery of antimicrobial compounds targeting bacterial type FAD synthetases.

PubMed

Sebastián, María; Anoz-Carbonell, Ernesto; Gracia, Begoña; Cossio, Pilar; Aínsa, José Antonio; Lans, Isaías; Medina, Milagros

2018-12-01

The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammoniagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also evaluated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties.

Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor.

PubMed

Samanta, Dipanjan; Widom, Joanne; Borbat, Peter P; Freed, Jack H; Crane, Brian R

2016-12-09

Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

FadA5 a thiolase from Mycobacterium tuberculosis - a unique steroid-binding pocket reveals the potential for drug development against tuberculosis

PubMed Central

Schaefer, Christin M.; Lu, Rui; Nesbitt, Natasha M.; Schiebel, Johannes; Sampson, Nicole S.; Kisker, Caroline

2014-01-01

Summary With the exception of HIV, tuberculosis (TB) is the leading cause of mortality among infectious diseases. The urgent need to develop new anti-tubercular drugs is apparent due to the increasing number of drug resistant Mycobacterium tuberculosis (Mtb) strains. Proteins involved in cholesterol import and metabolism have recently been discovered as potent targets against TB. FadA5, a thiolase from Mtb, is catalyzing the last step of the β-oxidation reaction of the cholesterol side-chain degradation under release of critical metabolites and was shown to be of importance during the chronic stage of TB infections. To gain structural and mechanistic insight on FadA5 we characterized the enzyme in different stages of the cleavage reaction and with a steroid bound to the binding pocket. Structural comparisons to human thiolases revealed that it should be possible to target FadA5 specifically and the steroid-bound structure provides a solid basis for the development of inhibitors against FadA5. PMID:25482540

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

PubMed Central

Xie, Q W; Leung, M; Fuortes, M; Sassa, S; Nathan, C

1996-01-01

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:8643499

Hydrogen Bond Switching among Flavin and Amino Acid Side Chains in the BLUF Photoreceptor Observed by Ultrafast Infrared Spectroscopy

PubMed Central

Bonetti, Cosimo; Mathes, Tilo; van Stokkum, Ivo H. M.; Mullen, Katharine M.; Groot, Marie-Louise; van Grondelle, Rienk; Hegemann, Peter; Kennis, John T. M.

2008-01-01

BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD•− and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD•− to result in FADH• on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH• C=N stretch marker mode, with tyrosine as the likely proton donor. FADH• is reoxidized in 67 ps (180 ps in D2O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by ∼180° through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch. PMID:18708458

Obesity resistance and deregulation of lipogenesis in Δ6-fatty acid desaturase (FADS2) deficiency.

PubMed

Stoffel, Wilhelm; Hammels, Ina; Jenke, Britta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Odenthal, Margarete; Thevis, Mario

2014-01-01

Δ-6-fatty acid desaturase (FADS2) is the key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs), the essential structural determinants of mammalian membrane lipid-bilayers. We developed the auxotrophic fads2(-/-) mouse mutant to assess the enigmatic role of ω3- and ω6-PUFAs in lipid homeostasis, membrane structure and function. Obesity resistance is another major phenotype of the fads2(-/-) mutant, the molecular basis of which is unknown. Phospholipidomic profiling of membrane systems of fads2(-/-)mice revealed diacylglycerol-structures, deprived of PUFAs but substituted with surrogate eicosa-5,11,14-trienoic acid. ω6-Arachidonic (AA) and ω3-docosahexaenoic acid (DHA) supplemented diets transformed fads2(-/-) into AA-fads2(-/-) and DHA-fads2(-/-) mutants. Severely altered phospholipid-bilayer structures of subcellular membranes of fads2(-/-) liver specifically interfered with maturation of transcription factor sterol-regulatory-element-binding protein, the key regulator of lipogenesis and lipid homeostasis. This study strengthens the concept that specific PUFA-substituted membrane phospholipid species are critical constituents of the structural platform operative in lipid homeostasis in normal and disease conditions.

Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

PubMed

Ochiai, Natsuki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

2016-05-01

Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level.

Insights on the structural perturbations in human MTHFR Ala222Val mutant by protein modeling and molecular dynamics.

PubMed

Abhinand, P A; Shaikh, Faraz; Bhakat, Soumendranath; Radadiya, Ashish; Bhaskar, L V K S; Shah, Anamik; Ragunath, P K

2016-01-01

Methylenetetrahydrofolate reductase (MTHFR) protein catalyzes the only biochemical reaction which produces methyltetrahydrofolate, the active form of folic acid essential for several molecular functions. The Ala222Val polymorphism of human MTHFR encodes a thermolabile protein associated with increased risk of neural tube defects and cardiovascular disease. Experimental studies have shown that the mutation does not affect the kinetic properties of MTHFR, but inactivates the protein by increasing flavin adenine dinucleotide (FAD) loss. The lack of completely solved crystal structure of MTHFR is an impediment in understanding the structural perturbations caused by the Ala222Val mutation; computational modeling provides a suitable alternative. The three-dimensional structure of human MTHFR protein was obtained through homology modeling, by taking the MTHFR structures from Escherichia coli and Thermus thermophilus as templates. Subsequently, the modeled structure was docked with FAD using Glide, which revealed a very good binding affinity, authenticated by a Glide XP score of -10.3983 (kcal mol(-1)). The MTHFR was mutated by changing Alanine 222 to Valine. The wild-type MTHFR-FAD complex and the Ala222Val mutant MTHFR-FAD complex were subjected to molecular dynamics simulation over 50 ns period. The average difference in backbone root mean square deviation (RMSD) between wild and mutant variant was found to be ~.11 Å. The greater degree of fluctuations in the mutant protein translates to increased conformational stability as a result of mutation. The FAD-binding ability of the mutant MTHFR was also found to be significantly lowered as a result of decreased protein grip caused by increased conformational flexibility. The study provides insights into the Ala222Val mutation of human MTHFR that induces major conformational changes in the tertiary structure, causing a significant reduction in the FAD-binding affinity.

Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.

PubMed

Chowdhury, Nilanjan Pal; Mowafy, Amr M; Demmer, Julius K; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

2014-02-21

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

PubMed Central

Gadave, Kaustubh S.; Panda, Santanu; Singh, Surender; Kalra, Shalini; Malakar, Dhruba; Mohanty, Ashok K.; Kaushik, Jai K.

2014-01-01

Background Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species. Methods and Findings Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m) of 0.11 sec−1 µM−1 of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M−1 cm−1 suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD+ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum. Conclusion A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way. PMID:24498153

A comparative study of dietary curcumin, nanocurcumin, and other classical amyloid-binding dyes for labeling and imaging of amyloid plaques in brain tissue of 5×-familial Alzheimer's disease mice.

PubMed

Maiti, Panchanan; Hall, Tia C; Paladugu, Leela; Kolli, Nivya; Learman, Cameron; Rossignol, Julien; Dunbar, Gary L

2016-11-01

Deposition of amyloid beta protein (Aβ) is a key component in the pathogenesis of Alzheimer's disease (AD). As an anti-amyloid natural polyphenol, curcumin (Cur) has been used as a therapy for AD. Its fluorescent activity, preferential binding to Aβ, as well as structural similarities with other traditional amyloid-binding dyes, make it a promising candidate for labeling and imaging of Aβ plaques in vivo. The present study was designed to test whether dietary Cur and nanocurcumin (NC) provide more sensitivity for labeling and imaging of Aβ plaques in brain tissues from the 5×-familial AD (5×FAD) mice than the classical Aβ-binding dyes, such as Congo red and Thioflavin-S. These comparisons were made in postmortem brain tissues from the 5×FAD mice. We observed that Cur and NC labeled Aβ plaques to the same degree as Aβ-specific antibody and to a greater extent than those of the classical amyloid-binding dyes. Cur and NC also labeled Aβ plaques in 5×FAD brain tissues when injected intraperitoneally. Nanomolar concentrations of Cur or NC are sufficient for labeling and imaging of Aβ plaques in 5×FAD brain tissue. Cur and NC also labeled different types of Aβ plaques, including core, neuritic, diffuse, and burned-out, to a greater degree than other amyloid-binding dyes. Therefore, Cur and or NC can be used as an alternative to Aβ-specific antibody for labeling and imaging of Aβ plaques ex vivo and in vivo. It can provide an easy and inexpensive means of detecting Aβ-plaque load in postmortem brain tissue of animal models of AD after anti-amyloid therapy.

FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

PubMed

Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

2017-04-11

The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi

2003-11-01

Electron-transferring flavoprotein (ETF), its redox partner flavoproteins, i.e., D-lactate dehydrogenase and butyryl-CoA dehydrogenase, and another well-known flavoprotein, flavodoxin, were purified from the same starting cell paste of an anaerobic bacterium, Megasphaera elsdenii. The purified ETF contained one mol FAD/mol ETF as the sole non-protein component and bound almost one mol of additional FAD. This preparation is a better subject for investigations of M. elsdenii ETF than the previously isolated ETF, which contains varying amounts of FAD and varying percentages of modified flavins such as 6-OH-FAD and 8-OH-FAD. The additionally bound FAD shows an anomalous absorption spectrum with strong absorption around 400 nm. This spectral change is not due to a chemical modification of the flavin ring because the flavin released by KBr or guanidine hydrochloride is normal FAD. It is also not due to unknown small molecules because the same spectrum appears when ETF is reconstituted from its guanidine-denatured subunits and FAD. A similar anomalous spectrum was observed for AMP-free pig ETF under acidic conditions, suggesting a common flavin environment between pig and M. elsdenii ETFs.

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

NASA Astrophysics Data System (ADS)

Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

2017-03-01

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

PubMed Central

Giancaspero, Teresa A.; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M.; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

2015-01-01

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD “chaperone.” The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.).

PubMed

Lee, Kyeong-Ryeol; Kim, Sun Hee; Go, Young-Sam; Jung, Sung Min; Roh, Kyung Hee; Kim, Jong-Bum; Suh, Mi-Chung; Lee, Sukchan; Kim, Hyun Uk

2012-11-10

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60-70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion. Copyright © 2012 Elsevier B.V. All rights reserved.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans*

PubMed Central

Chowdhury, Nilanjan Pal; Mowafy, Amr M.; Demmer, Julius K.; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

2014-01-01

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD+ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH− is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH− by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD⨪, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH− that converts crotonyl-CoA to butyryl-CoA. PMID:24379410

Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.

PubMed

Yokom, Adam L; Morishima, Yoshihiro; Lau, Miranda; Su, Min; Glukhova, Alisa; Osawa, Yoichi; Southworth, Daniel R

2014-06-13

Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal Structure of FadA Adhesin from Fusobacterium nucleatum Reveals a Novel Oligomerization Motif, the Leucine Chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori

2009-04-07

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 {angstrom} crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel {alpha}-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tailmore » association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the 'leucine chain.' The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.« less

Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2*

PubMed Central

Leung, Kevin K. K.; Shilton, Brian H.

2013-01-01

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P212121 to P21, with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4–5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe126–Leu136) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch. PMID:23471972

The RFK catalytic cycle of the pathogen Streptococcus pneumoniae shows species-specific features in prokaryotic FMN synthesis.

PubMed

Sebastián, María; Velázquez-Campoy, Adrián; Medina, Milagros

2018-12-01

Emergence of multidrug-resistant bacteria forces us to explore new therapeutic strategies, and proteins involved in key metabolic pathways are promising anti-bacterial targets. Bifunctional flavin-adenine dinucleotide (FAD) synthetases (FADS) are prokaryotic enzymes that synthesise the flavin mononucleotide (FMN) and FAD cofactors. The FADS from the human pathogen Streptococcus pneumoniae (SpnFADS)-causative agent of pneumonia in humans - shows relevant catalytic dissimilarities compared to other FADSs. Here, by integrating thermodynamic and kinetic data, we present a global description of the riboflavin kinase activity of SpnFADS, as well as of the inhibition mechanisms regulating this activity. Our data shed light on biophysical determinants that modulate species-specific conformational changes leading to catalytically competent conformations, as well as binding rates and affinities of substrates versus products. This knowledge paves the way for the development of tools - that taking advantage of the regulatory dissimilarities during FMN biosynthesis in different species - might be used in the discovery of specific anti-pneumococcal drugs.

Molecular Characterization and Expression of a Novel Alcohol Oxidase from Aspergillus terreus MTCC6324

PubMed Central

Chakraborty, Mitun; Goel, Manish; Chinnadayyala, Somasekhar R.; Dahiya, Ujjwal Ranjan; Ghosh, Siddhartha Sankar; Goswami, Pranab

2014-01-01

The alcohol oxidase (AOx) cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g−1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an ab-initio protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (kcat/Km) of 7829.5 min−1 mM−1 for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application. PMID:24752075

Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324.

PubMed

Chakraborty, Mitun; Goel, Manish; Chinnadayyala, Somasekhar R; Dahiya, Ujjwal Ranjan; Ghosh, Siddhartha Sankar; Goswami, Pranab

2014-01-01

The alcohol oxidase (AOx) cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g-1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an ab-initio protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (kcat/Km) of 7829.5 min-1 mM-1 for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.

Type II flavohemoglobin of Mycobacterium smegmatis oxidizes d-lactate and mediate electron transfer.

PubMed

Thakur, Naveen; Kumar, Ashwani; Dikshit, Kanak L

2018-06-01

Two distantly related flavohemoglobins (FHbs), MsFHbI and MsFHbII, having crucial differences in their heme and reductase domains, co-exist in Mycobacterium smegmatis. Function of MsFHbI is associated with nitric-oxide detoxification but physiological relevance of MsFHbII remains unknown. This study unravels some unique spectral and functional characteristics of MsFHbII. Unlike conventional type I FHbs, MsFHbII lacks nitric-oxide dioxygenase and NADH oxidase activities but utilizes d-lactate as an electron donor to mediate electron transfer. MsFHbII carries a d-lactate dehydrogenase type FAD binding motif in its reductase domain and oxidizes d-lactate in a FAD dependent manner to reduce the heme iron, suggesting that the globin is acting as an electron acceptor. Importantly, expression of MsFHbII in Escherichia coli imparted protection under oxidative stress, suggesting its important role in stress management of its host. Since M. smegmatis lacks the gene encoding for d-lactate dehydrogenase and d-lactate is produced during aerobic metabolism and also as a by-product of lipid peroxidation, the ability of MsFHbII to metabolize d-lactate may provide it a unique ability to balance the oxidative stress generated due to accumulation of d-lactate in the cell and at the same time sequester electrons and pass it to the respiratory apparatus. Copyright © 2018 Elsevier B.V. All rights reserved.

Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein

PubMed Central

Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

2015-01-01

ABSTRACT The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. PMID:25944861

Exploring the Landscape of Modern Academic Psychology: Finding and Filling the Holes

ERIC Educational Resources Information Center

Rozin, Paul

2007-01-01

Like any other domain of human activity, psychology has its fads and fashions. One consequence of fads is an overconcentration of resources on specific problems or approaches, which leaves other important problems or approaches (holes) underappreciated and understudied. This article is primarily about different factors (such as negativity bias,…

Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33.

PubMed

Vergnolle, Olivia; Xu, Hua; Blanchard, John S

2013-09-27

Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.

Omega-3 fatty acid desaturase gene family from two ω-3 sources, Salvia hispanica and Perilla frutescens: Cloning, characterization and expression

PubMed Central

Xue, Yufei; Chen, Baojun; Win, Aung Naing; Fu, Chun; Lian, Jianping; Liu, Xue; Wang, Rui; Zhang, Xingcui

2018-01-01

Omega-3 fatty acid desaturase (ω-3 FAD, D15D) is a key enzyme for α-linolenic acid (ALA) biosynthesis. Both chia (Salvia hispanica) and perilla (Perilla frutescens) contain high levels of ALA in seeds. In this study, the ω-3 FAD gene family was systematically and comparatively cloned from chia and perilla. Perilla FAD3, FAD7, FAD8 and chia FAD7 are encoded by single-copy (but heterozygous) genes, while chia FAD3 is encoded by 2 distinct genes. Only 1 chia FAD8 sequence was isolated. In these genes, there are 1 to 6 transcription start sites, 1 to 8 poly(A) tailing sites, and 7 introns. The 5’UTRs of PfFAD8a/b contain 1 to 2 purine-stretches and 2 pyrimidine-stretches. An alternative splice variant of ShFAD7a/b comprises a 5’UTR intron. Their encoded proteins harbor an FA_desaturase conserved domain together with 4 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis validated their identity of dicot microsomal or plastidial ω-3 FAD proteins, and revealed some important evolutionary features of plant ω-3 FAD genes such as convergent evolution across different phylums, single-copy status in algae, and duplication events in certain taxa. The qRT-PCR assay showed that the ω-3 FAD genes of two species were expressed at different levels in various organs, and they also responded to multiple stress treatments. The functionality of the ShFAD3 and PfFAD3 enzymes was confirmed by yeast expression. The systemic molecular and functional features of the ω-3 FAD gene family from chia and perilla revealed in this study will facilitate their use in future studies on genetic improvement of ALA traits in oilseed crops. PMID:29351555

Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

NASA Astrophysics Data System (ADS)

Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

2015-04-01

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

PubMed Central

Augustin, Peter; Toplak, Marina; Fuchs, Katharina; Gerstmann, Eva Christine; Prassl, Ruth; Winkler, Andreas; Macheroux, Peter

2018-01-01

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria. PMID:29301933

Detailed molecular analyses of the hexon loop-1 and fibers of fowl aviadenoviruses reveal new insights into the antigenic relationship and confirm that specific genotypes are involved in field outbreaks of inclusion body hepatitis.

PubMed

Schachner, Anna; Marek, Ana; Grafl, Beatrice; Hess, Michael

2016-04-15

Forty-eight fowl aviadenoviruses (FAdVs) isolated from recent IBH outbreaks across Europe were investigated, by utilizing for the first time the two major adenoviral antigenic domains, hexon loop-1 and fiber, for compound molecular characterization of IBH-associated FAdVs. Successful target gene amplification, following virus isolation in cell culture or from FTA-card samples, demonstrated presence of FAdVs in all cases indicative for IBH. Based on hexon loop-1 analysis, 31 European field isolates exhibited highest nucleotide identity (>97.2%) to reference strains FAdV-2 or -11 representing FAdV-D, while 16 and one European isolates shared >96.0% nucleotide identity with FAdV-8a and -8b, or FAdV-7, the prototype strains representing FAdV-E. These results extend recognition of specific FAdV-D and FAdV-E affiliate genotypes as causative agents of IBH to the European continent. In all isolates, species specificity determined by fiber gene analysis correlated with hexon-based typing. A threshold of 72.0% intraspecies nucleotide identity between fibers from investigated prototype and field strains corresponded with demarcation criteria proposed for hexon, suggesting fiber-based analysis as a complementary tool for molecular FAdV typing. A limited number of strains exhibited inconsistencies between hexon and fiber subclustering, indicating potential constraints for single-gene based typing of those FAdVs. Within FAdV-D, field isolate fibers shared a high degree of nucleotide (>96.7%) and aa (>95.8%) identity, while FAdV-E field isolate fibers displayed greater nucleotide divergence of up to 22.6%, resulting in lower aa identities of >81.7%. Furthermore, comparison with FAdVs from IBH outbreaks outside Europe revealed close genetic relationship in the fiber, independent of the strains' geographic origin. Copyright © 2016 Elsevier B.V. All rights reserved.

Stabilization of non-productive conformations underpins rapid electron transfer to electron-transferring flavoprotein.

PubMed

Toogood, Helen S; van Thiel, Adam; Scrutton, Nigel S; Leys, David

2005-08-26

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.

Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

NASA Astrophysics Data System (ADS)

Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

2005-08-01

The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl) and a small non-interacting conformation (BLUF nc). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

DOE PAGES

Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; ...

2015-03-23

N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producingmore » more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.« less

The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength

PubMed Central

Campelo, Diana; Lautier, Thomas; Urban, Philippe; Esteves, Francisco; Bozonnet, Sophie; Truan, Gilles; Kranendonk, Michel

2017-01-01

NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners. PMID:29163152

Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jian, E-mail: jian@cfs.bioment.umaryland.edu; Fu, Yi; Li, Ge

2012-08-31

Highlights: Black-Right-Pointing-Pointer Metal nanoparticle for fluorescence cell imaging. Black-Right-Pointing-Pointer Non-invasive emission detection of coenzyme in cell on time-resolved confocal microscope. Black-Right-Pointing-Pointer Near-field interaction of flavin adenine dinucleotide with silver substrate. Black-Right-Pointing-Pointer Isolation of emissions by coenzymes from cellular autofluorescence on fluorescence cell imaging. -- Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent,more » but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.« less

Detection of Fusobacterium Nucleatum and fadA Adhesin Gene in Patients with Orthodontic Gingivitis and Non-Orthodontic Periodontal Inflammation

PubMed Central

Wang, Jianning; Guo, Yang; Zhang, Yujie; Xiao, Shuiqing

2014-01-01

Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p<0.01) while it was higher in orthodontic gingivitis group than in health people (p<0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis. PMID:24416378

Detection of fusobacterium nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation.

PubMed

Liu, Ping; Liu, Yi; Wang, Jianning; Guo, Yang; Zhang, Yujie; Xiao, Shuiqing

2014-01-01

Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p<0.01) while it was higher in orthodontic gingivitis group than in health people (p<0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis.

RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides.

PubMed

Liu, Su; Liang, Qing-Mei; Zhou, Wen-Wu; Jiang, Yan-Dong; Zhu, Qing-Zi; Yu, Hang; Zhang, Chuan-Xi; Gurr, Geoff M; Zhu, Zeng-Rong

2015-01-01

NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens. © 2014 Society of Chemical Industry.

Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

PubMed Central

Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

2015-01-01

NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

Associations between variants of FADS genes and omega-3 and omega-6 milk fatty acids of Canadian Holstein cows

PubMed Central

2014-01-01

Background Fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes code respectively for the enzymes delta-5 and delta-6 desaturases which are rate limiting enzymes in the synthesis of polyunsaturated omega-3 and omega-6 fatty acids (FAs). Omega-3 and-6 FAs as well as conjugated linoleic acid (CLA) are present in bovine milk and have demonstrated positive health effects in humans. Studies in humans have shown significant relationships between genetic variants in FADS1 and 2 genes with plasma and tissue concentrations of omega-3 and-6 FAs. The aim of this study was to evaluate the extent of sequence variations within these two genes in Canadian Holstein cows as well as the association between sequence variants and health promoting FAs in milk. Results Thirty three SNPs were detected within the studied regions of genes including a synonymous mutation (FADS1-07, rs42187261, 306Tyr > Tyr) in exon 8 of FADS1, a non-synonymous mutation (FADS2-14, rs211580559, 294Ala > Val) within FADS2 exon 7, a splice site SNP (FADS2-05, rs211263660), a 3′UTR SNP (FADS2-23, rs109772589), and another 3′UTR SNP with an effect on a microRNA binding site within FADS2 gene (FADS2-19, rs210169303). Association analyses showed significant relations between three out of seven tested SNPs and several FAs. Significant associations (FDR P < 0.05) were recorded between FADS2-23 (rs109772589) and two omega-6 FAs (dihomogamma linolenic acid [C20:3n6] and arachidonic acid [C20:4n6]), FADS1-07 (rs42187261) and one omega-3 FA (eicosapentaenoic acid, C20:5n3) and tricosanoic acid (C23:0), and one intronic SNP, FADS1-01 (rs136261927) and C20:3n6. Conclusion Our study has demonstrated positive associations between three SNPs within FADS1 and FADS2 genes (a SNP within the 3’UTR, a synonymous SNP and an intronic SNP), with three milk PUFAs of Canadian Holstein cows thus suggesting possible involvement of synonymous and non-coding region variants in FA synthesis. These SNPs may serve as potential genetic markers in breeding programs to increase milk FAs that are of benefit to human health. PMID:24533445

Associations between variants of FADS genes and omega-3 and omega-6 milk fatty acids of Canadian Holstein cows.

PubMed

Ibeagha-Awemu, Eveline M; Akwanji, Kingsley A; Beaudoin, Frédéric; Zhao, Xin

2014-02-17

Fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes code respectively for the enzymes delta-5 and delta-6 desaturases which are rate limiting enzymes in the synthesis of polyunsaturated omega-3 and omega-6 fatty acids (FAs). Omega-3 and-6 FAs as well as conjugated linoleic acid (CLA) are present in bovine milk and have demonstrated positive health effects in humans. Studies in humans have shown significant relationships between genetic variants in FADS1 and 2 genes with plasma and tissue concentrations of omega-3 and-6 FAs. The aim of this study was to evaluate the extent of sequence variations within these two genes in Canadian Holstein cows as well as the association between sequence variants and health promoting FAs in milk. Thirty three SNPs were detected within the studied regions of genes including a synonymous mutation (FADS1-07, rs42187261, 306Tyr > Tyr) in exon 8 of FADS1, a non-synonymous mutation (FADS2-14, rs211580559, 294Ala > Val) within FADS2 exon 7, a splice site SNP (FADS2-05, rs211263660), a 3'UTR SNP (FADS2-23, rs109772589), and another 3'UTR SNP with an effect on a microRNA binding site within FADS2 gene (FADS2-19, rs210169303). Association analyses showed significant relations between three out of seven tested SNPs and several FAs. Significant associations (FDR P < 0.05) were recorded between FADS2-23 (rs109772589) and two omega-6 FAs (dihomogamma linolenic acid [C20:3n6] and arachidonic acid [C20:4n6]), FADS1-07 (rs42187261) and one omega-3 FA (eicosapentaenoic acid, C20:5n3) and tricosanoic acid (C23:0), and one intronic SNP, FADS1-01 (rs136261927) and C20:3n6. Our study has demonstrated positive associations between three SNPs within FADS1 and FADS2 genes (a SNP within the 3'UTR, a synonymous SNP and an intronic SNP), with three milk PUFAs of Canadian Holstein cows thus suggesting possible involvement of synonymous and non-coding region variants in FA synthesis. These SNPs may serve as potential genetic markers in breeding programs to increase milk FAs that are of benefit to human health.

Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

DOE PAGES

Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; ...

2015-05-05

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs

PubMed Central

Surm, Joachim M.; Prentis, Peter J.; Pavasovic, Ana

2015-01-01

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs. PMID:26308548

Familial Alzheimer's disease mutations in presenilin 1 do not alter levels of the secreted amyloid-beta protein precursor generated by beta-secretase cleavage.

PubMed

Zhang, Can; Browne, Andrew; Kim, Doo Yeon; Tanzi, Rudolph E

2010-02-01

Alzheimer's disease (AD) is an insidious and progressive disease with a genetically complex and heterogenous etiology. More than 200 fully penetrant mutations in the amyloid beta-protein precursor (APP), presenilin 1 (or PSEN1), and presenilin 2 (PSEN2) have been linked to early-onset familial AD (FAD). 177 PSEN1 FAD mutations have been identified so far and account for more than approximately 80% of all FAD mutations. All PSEN1 FAD mutations can increase the Abeta42:Abeta40 ratio with seemingly different and incompletely understood mechanisms. A recent study has shown that the 286 amino acid N-terminal fragment of APP (N-APP), a proteolytic product of beta-secretase-derived secreted form of APP (sAPPbeta), could bind the death receptor, DR6, and lead to neurodegeneration. Here we asked whether PSEN1 FAD mutations lead to neurodegeneration by modulating sAPPbeta levels. All four different PSEN1 FAD mutations tested (in three mammalian cell lines) did not alter sAPPbeta levels. Therefore PS1 mutations do not appear to contribute to AD pathogenesis via altered production of sAPPbeta.

SIRT3 and SIRT5 Regulate the Enzyme Activity and Cardiolipin Binding of Very Long-Chain Acyl-CoA Dehydrogenase

PubMed Central

Zhang, Yuxun; Bharathi, Sivakama S.; Rardin, Matthew J.; Uppala, Radha; Verdin, Eric; Gibson, Bradford W.; Goetzman, Eric S.

2015-01-01

SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane. PMID:25811481

Comparative study of flavins binding with human serum albumin: a fluorometric, thermodynamic, and molecular dynamics approach.

PubMed

Sengupta, Abhigyan; Sasikala, Wilbee D; Mukherjee, Arnab; Hazra, Partha

2012-06-04

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brautigam, Chad A.; Chuang, Jacinta L.; Tomchick, Diana R.

Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial {alpha}-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: noncovalently bound FAD and a transiently bound substrate, NAD{sup +}. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD{sup +} or NADH have been determined at resolutions of 2.5 {angstrom} and 2.1 {angstrom}, respectively. Although the overall fold of the enzyme is similarmore » to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD{sup +} bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD{sup +} or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD{sup +}-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.« less

Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armstrong, G.A.; Hearst, J.E.; Alberti, M.

1990-12-01

Carotenoids comprise one of the most widespread classes of pigments found in nature. The first reactions of C{sub 40} carotenoid biosynthesis proceed through common intermediates in all organisms, suggesting the evolutionary conservation of early enzymes from this pathway. The authors report here the nucleotide sequence of three genes from the carotenoid biosynthesis gene cluster of Erwinia herbicola, a nonphotosynthetic epiphytic bacterium, which encode homologs of the CrtB, CrtE, and CrtI proteins of Rhodobacter capsulatus, a purple nonsulfur photosynthetic bacterium. CrtB (prephytoene pyrophosphate synthase), CrtE (phytoene synthase), and CrtI (phytoene dehydrogenase) are required for the first three reactions specific to themore » carotenoid branch of general isoprenoid metabolism. All three dehydrogenases possess a hydrophobic N-terminal domain containing a putative ADP-binding {beta}{alpha}{beta} fold characteristic of enzymes known to bind FAD or NAD(P) cofactors. These data indicate the structural conservation of early carotenoid biosynthesis enzymes in evolutionary diverse organisms.« less

Visual short-term memory binding deficit in familial Alzheimer's disease.

PubMed

Liang, Yuying; Pertzov, Yoni; Nicholas, Jennifer M; Henley, Susie M D; Crutch, Sebastian; Woodward, Felix; Leung, Kelvin; Fox, Nick C; Husain, Masud

2016-05-01

Long-term episodic memory deficits in Alzheimer's disease (AD) are well characterised but, until recently, short-term memory (STM) function has attracted far less attention. We employed a recently-developed, delayed reproduction task which requires participants to reproduce precisely the remembered location of items they had seen only seconds previously. This paradigm provides not only a continuous measure of localization error in memory, but also an index of relational binding by determining the frequency with which an object is misplaced to the location of one of the other items held in memory. Such binding errors in STM have previously been found on this task to be sensitive to medial temporal lobe (MTL) damage in focal lesion cases. Twenty individuals with pathological mutations in presenilin 1 or amyloid precursor protein genes for familial Alzheimer's disease (FAD) were tested together with 62 healthy controls. Participants were assessed using the delayed reproduction memory task, a standard neuropsychological battery and structural MRI. Overall, FAD mutation carriers were worse than controls for object identity as well as in gross localization memory performance. Moreover, they showed greater misbinding of object identity and location than healthy controls. Thus they would often mislocalize a correctly-identified item to the location of one of the other items held in memory. Significantly, asymptomatic gene carriers - who performed similarly to healthy controls on standard neuropsychological tests - had a specific impairment in object-location binding, despite intact memory for object identity and location. Consistent with the hypothesis that the hippocampus is critically involved in relational binding regardless of memory duration, decreased hippocampal volume across FAD participants was significantly associated with deficits in object-location binding but not with recall precision for object identity or localization. Object-location binding may therefore provide a sensitive cognitive biomarker for MTL dysfunction in a range of diseases including AD. Copyright © 2016. Published by Elsevier Ltd.

Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor.

PubMed

Ichinose, H; Wariishi, H; Tanaka, H

2002-09-01

A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.

Reassessment of the Genetic Regulation of Fatty Acid Synthesis in Escherichia coli: Global Positive Control by the Dual Functional Regulator FadR

PubMed Central

My, L.; Ghandour Achkar, N.; Viala, J. P.

2015-01-01

ABSTRACT In Escherichia coli, the FadR transcriptional regulator represses the expression of fatty acid degradation (fad) genes. However, FadR is also an activator of the expression of fabA and fabB, two genes involved in unsaturated fatty acid synthesis. Therefore, FadR plays an important role in maintaining the balance between saturated and unsaturated fatty acids in the membrane. We recently showed that FadR also activates the promoter upstream of the fabH gene (L. My, B. Rekoske, J. J. Lemke, J. P. Viala, R. L. Gourse, and E. Bouveret, J Bacteriol 195:3784–3795, 2013, doi:10.1128/JB.00384-13). Furthermore, recent transcriptomic and proteomic data suggested that FadR activates the majority of fatty acid (FA) synthesis genes. In the present study, we tested the role of FadR in the expression of all genes involved in FA synthesis. We found that FadR activates the transcription of all tested FA synthesis genes, and we identified the FadR binding site for each of these genes. This necessitated the reassessment of the transcription start sites for accA and accB genes described previously, and we provide evidence for the presence of multiple promoters driving the expression of these genes. We showed further that regulation by FadR impacts the amount of FA synthesis enzymes in the cell. Our results show that FadR is a global regulator of FA metabolism in E. coli, acting both as a repressor of catabolism and an activator of anabolism, two directly opposing pathways. IMPORTANCE In most bacteria, a transcriptional regulator tunes the level of FA synthesis enzymes. Oddly, such a global regulator still was missing for E. coli, which nonetheless is one of the prominent model bacteria used for engineering biofuel production using the FA synthesis pathway. Our work identifies the FadR functional dual regulator as a global activator of almost all FA synthesis genes in E. coli. Because FadR also is the repressor of FA degradation, FadR acts both as a repressor and an activator of the two opposite pathways of FA degradation and synthesis. Our results show that there are still discoveries waiting to be made in the understanding of the genetic regulation of FA synthesis, even in the very well-known bacterium E. coli. PMID:25802297

Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change1[W][OA

PubMed Central

Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

2010-01-01

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate. PMID:20460582

Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.

PubMed

Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

2010-07-01

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.

From Cholesterogenesis to Steroidogenesis: Role of Riboflavin and Flavoenzymes in the Biosynthesis of Vitamin D12

PubMed Central

Pinto, John T.; Cooper, Arthur J. L.

2014-01-01

Flavin-dependent monooxygenases and oxidoreductases are located at critical branch points in the biosynthesis and metabolism of cholesterol and vitamin D. These flavoproteins function as obligatory intermediates that accept 2 electrons from NAD(P)H with subsequent 1-electron transfers to a variety of cytochrome P450 (CYP) heme proteins within the mitochondria matrix (type I) and the (microsomal) endoplasmic reticulum (type II). The mode of electron transfer in these systems differs slightly in the number and form of the flavin prosthetic moiety. In the type I mitochondrial system, FAD-adrenodoxin reductase interfaces with adrenodoxin before electron transfer to CYP heme proteins. In the microsomal type II system, a diflavin (FAD/FMN)-dependent cytochrome P450 oxidoreductase [NAD(P)H-cytochrome P450 reductase (CPR)] donates electrons to a multitude of heme oxygenases. Both flavoenzyme complexes exhibit a commonality of function with all CYP enzymes and are crucial for maintaining a balance of cholesterol and vitamin D metabolites. Deficits in riboflavin availability, imbalances in the intracellular ratio of FAD to FMN, and mutations that affect flavin binding domains and/or interactions with client proteins result in marked structural alterations within the skeletal and central nervous systems similar to those of disorders (inborn errors) in the biosynthetic pathways that lead to cholesterol, steroid hormones, and vitamin D and their metabolites. Studies of riboflavin deficiency during embryonic development demonstrate congenital malformations similar to those associated with genetic alterations of the flavoenzymes in these pathways. Overall, a deeper understanding of the role of riboflavin in these pathways may prove essential to targeted therapeutic designs aimed at cholesterol and vitamin D metabolism. PMID:24618756

Conjugated Fatty Acid Synthesis

PubMed Central

Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

2012-01-01

Conjugated linolenic acids (CLNs), 18:3 Δ9,11,13, lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ9,12,15). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ9cis,11trans,13cis) or α-eleostearic acid (18:3 Δ9cis,11trans,13trans). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation. PMID:22451660

Positional cloning of the chromosome 14 Alzheimer`s disease locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, R.F.; Korenblat, K.M.; Goate, A.M.

1994-09-01

Genetic linkage analysis had indicated a locus for familial early-onset Alzheimer`s disease (FAD) on chromosome 14 at q24.3. The FAD locus has been shown previously to lie between the dinucleotide markers D14S61 and D14S63, a genetic distance of approximately 13 cM. We are currently attempting to identify the gene using a positional cloning strategy. The first step towards the isolation and characterization of this locus was the construction of an overlapping YAC contig covering the entire region. Over forty YACs which map to this region have been isolated from the St. Louis and CEPH libraries by a combination of YACmore » end sequence walking and sequence tagged site mapping. Our contig fully spans the complete domain, encompassing all genetic markers non-recombinant with FAD (i.e. D14S76, D14S43, D14S71, D14S77) and the two nearest flanking FAD-recombinant markers. With restriction mapping of the domain, we can determine the exact size of the region. As a second step, the YACs in this contig are currently being inspected for expressed sequences by exon trapping, initially on those YACs known to be nonchimeric. We have currently made exon-trapped libraries from YACs that have the markers D14S76 and D14S43. Sequence analysis of these libraries indicates that a trapped exon is identified on average for each 30 kb of YAC DNA. The trapped exons are being screened to identify likely candidate genes, which will be examined for mutations in FAD families.« less

Desaturase and elongase-limiting endogenous long-chain polyunsaturated fatty acid biosynthesis.

PubMed

Zhang, Ji Yao; Kothapalli, Kumar S D; Brenna, J Thomas

2016-03-01

Endogenous synthesis of the long-chain polyunsaturated fatty acids (LCPUFAs) is mediated by the fatty acid desaturase (FADS) gene cluster (11q12-13.1) and elongation of very long-chain fatty acids 2 (ELOVL2) (6p24.2) and ELOVL5 (6p12.1). Although older biochemical work identified the product of one gene, FADS2, rate limiting for LCPUFA synthesis, recent studies suggest that polymorphisms in any of these genes can limit accumulation of product LCPUFA. Genome-wide association study (GWAS) of Greenland Inuit shows strong adaptation signals within FADS gene cluster, attributed to high omega-3 fatty acid intake, while GWAS found ELOVL2 associated with sleep duration, age and DNA methylation. ELOVL5 coding mutations cause spinocerebellar ataxia 38, and epigenetic marks were associated with depression and suicide risk. Two sterol response element binding sites were found on ELOVL5, a SREBP-1c target gene. Minor allele carriers of a 3 single nucleotide polymorphism (SNP) haplotype in ELOVL2 have decreased 22 : 6n-3 levels. Unequivocal molecular evidence shows mammalian FADS2 catalyzes direct Δ4-desaturation to yield 22 : 6n-3 and 22 : 5n-6. An SNP near FADS1 influences the levels of 5-lipoxygenase products and epigenetic alteration. Genetic polymorphisms within FADS and ELOVL can limit LCPUFA product accumulation at any step of the biosynthetic pathway.

The N-terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre.

PubMed

Hofhaus, Götz; Lee, Jeung-Eun; Tews, Ivo; Rosenberg, Beate; Lisowsky, Thomas

2003-04-01

Yeast Erv1p is a ubiquitous FAD-dependent sulfhydryl oxidase, located in the intermembrane space of mitochondria. The dimeric enzyme is essential for survival of the cell. Besides the redox-active CXXC motif close to the FAD, Erv1p harbours two additional cysteine pairs. Site-directed mutagenesis has identified all three cysteine pairs as essential for normal function. The C-terminal cysteine pair is of structural importance as it contributes to the correct arrangement of the FAD-binding fold. Variations in dimer formation and unique colour changes of mutant proteins argue in favour of an interaction between the N-terminal cysteine pair with the redox centre of the partner monomer.

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants▿

PubMed Central

Ruckenstuhl, Christoph; Lang, Silvia; Poschenel, Andrea; Eidenberger, Armin; Baral, Pravas Kumar; Kohút, Peter; Hapala, Ivan; Gruber, Karl; Turnowsky, Friederike

2007-01-01

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines. PMID:17043127

Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD.

PubMed

Xu, Lei; Wen, Bin; Wang, Yuan; Tian, Changqing; Wu, Mingcai; Zhu, Guoping

2017-06-19

Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as "site 377", that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light-dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides.

PubMed

Stokin, Gorazd B; Almenar-Queralt, Angels; Gunawardena, Shermali; Rodrigues, Elizabeth M; Falzone, Tomás; Kim, Jungsu; Lillo, Concepción; Mount, Stephanie L; Roberts, Elizabeth A; McGowan, Eileen; Williams, David S; Goldstein, Lawrence S B

2008-11-15

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer's disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-beta peptides (Abeta), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Abeta poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Abeta, we perturbed Abeta generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Abeta42/Abeta40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Abeta with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Abeta ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Abeta.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

DOE Office of Scientific and Technical Information (OSTI.GOV)

Begley, Darren W.; Davies, Douglas R.; Hartley, Robert C.

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting amore » fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.« less

Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters.

PubMed

Feng, Youjun; Cronan, John E

2011-04-01

Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription-translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non-specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT-PCR and fatty acid composition analyses demonstrated that V. cholerae FabR was a functional repressor of unsaturated fatty acid synthesis. However, in contrast to E. coli, gel mobility shift assays indicated that neither E. coli nor V. cholerae FabRs bound the V. cholerae fabB promoter, although both proteins efficiently bound the V. cholerae fabA promoter. This asymmetry was shown to be due to the lack of a FabR binding site within the V. cholerae fabB promoter region. © 2011 Blackwell Publishing Ltd.

Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches.

PubMed

Gobin-Limballe, Stéphanie; McAndrew, Ryan P; Djouadi, Fatima; Kim, Jung-Ja; Bastin, Jean

2010-05-01

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ss-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme-FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer-monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach. Copyright 2010 Elsevier B.V. All rights reserved.

Study of Proton Transfer in E. Coli Photolyase

NASA Astrophysics Data System (ADS)

Zhang, Meng; Liu, Zheyun; Li, Jiang; Wang, Lijuan; Zhong, Dongping

2013-06-01

Photolyase is a flavoprotein which utilizes blue-light energy to repair UV-light damaged DNA. The catalytic cofactor of photolyase, flavin adenine dinucleotide (FAD), has five redox states. Conversions between these redox states involve intraprotein electron transfer and proton transfer, which play important role in protein function. Here we systematically studied proton transfer in E. coli photolyase in vitro by site-directed mutagenesis and steady-state UV-vis spectroscopy, and proposed the proton channel in photolyase. We found that in the mutant N378C/E363L, proton channel was completely eliminated when DNA substrate was bound to the protein. Proton is suggested to be transported from protein surface to FAD by two pathways: the proton relay pathway through E363 and surface water to N378 and then to FAD; and the proton diffusion pathway through the substrate binding pocket. In addition, reaction kinetics of conversions between the redox states was then solved and redox potentials of the redox states were determined. These results described a complete picture of FAD redox changes, which are fundamental to the functions of all flavoenzymes.

Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1.

PubMed

Sokolova, Olga; Cecala, Christine; Gopal, Anand; Cortazar, Frank; McDowell-Buchanan, Carla; Sancar, Aziz; Gindt, Yvonne M; Schelvis, Johannes P M

2007-03-27

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.

Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein.

PubMed

Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V

2015-05-05

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete's periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg(2+)-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg(2+)-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp's dual activities, thereby underscoring the role of Mg(2+) in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. Treponema pallidum, the syphilis spirochete, exploits its periplasmic lipoproteins for a number of essential physiologic processes. One of these, flavin-trafficking protein (Ftp), not only exploits its catalytic center to mediate posttranslational flavinylation of proteins (to create flavoproteins) but also likely maintains the periplasmic flavin pool via its unique ability to hydrolyze FAD. This functional diversity within a single lipoprotein is quite remarkable and reflects the enzymatic versatility of the treponemal lipoproteins, as well as molecular parsimony in an organism with a limited genome. Ftp-mediated protein flavinylation in the periplasm also likely is a key aspect of a predicted flavin-dependent Rnf-based redox homeostasis system at the cytoplasmic membrane of T. pallidum. In addition to its importance in T. pallidum physiology, Ftp homologs exist in other bacteria, thereby expanding our understanding of the bacterial periplasm as a metabolically active subcellular compartment for flavoprotein biogenesis as well as flavin homeostasis. Copyright © 2015 Deka et al.

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes.

PubMed

Thorpe, Colin; Hoober, Karen L; Raje, Sonali; Glynn, Nicole M; Burnside, Joan; Turi, George K; Coppock, Donald L

2002-09-01

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.

Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Soo-Ik; Hammes, G.G.

1989-11-01

Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chickenmore » and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.« less

Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase.

PubMed

Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

2012-05-11

Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamill, Michael J.; Jost, Marco; Wong, Cintyu

2011-11-21

The process known as 'adaptive response' allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity formore » flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 {+-} 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 {angstrom} resolution crystal structure in space group P3{sub 2} that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.« less

Biochemical characterization of the selenoproteome in Gallus gallus via bioinformatics analysis: structure-function relationships and interactions of binding molecules.

PubMed

Zhu, Shi-Yong; Li, Xue-Nan; Sun, Xiao-Chen; Lin, Jia; Li, Wei; Zhang, Cong; Li, Jin-Long

2017-02-22

Knowledge about mammalian selenoproteins is increasing. However, the selenoproteome of birds remains considerably less understood, especially concerning its biochemical characterization, structure-function relationships and the interactions of binding molecules. In this work, the SECIS elements, subcellular localization, protein domains and interactions of binding molecules of the selenoproteome in Gallus gallus were analyzed using bioinformatics tools. We carried out comprehensive analyses of the structure-function relationships and interactions of the binding molecules of selenoproteins, to provide biochemical characterization of the selenoproteome in Gallus gallus. Our data provided a wealth of information on the biochemical functions of bird selenoproteins. Members of the selenoproteome were found to be involved in various biological processes in chickens, such as in antioxidants, maintenance of the redox balance, Se transport, and interactions with metals. Six membrane-bound selenoproteins (SelI, SelK, SelS, SelT, DIO1 and DIO3) played important roles in maintaining the membrane integrity. Chicken selenoproteins were classified according to their ligand binding sites as zinc-containing matrix metalloselenoproteins (Sep15, MsrB1, SelW and SelM), POP-containing selenoproteins (GPx1-4), FAD-interacting selenoproteins (TrxR1-3), secretory transport selenoproteins (GPx3 and SelPa) and other selenoproteins. The results of our study provided new evidence for the unknown biological functions of the selenoproteome in birds. Future research is required to confirm the novel biochemical functions of bird selenoproteins.

Structures of NADH and CH[subscript 3]-H[subscript 4] Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pejchal, Robert; Sargeant, Ryan; Ludwig, Martha L.

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH{sub 2}-H{sub 4}folate) to 5-methyltetrahydrofolate (CH{sub 3}-H{sub 4}folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a ({beta}{alpha}){sub 8} barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH{sub 3}-H{sub 4}folate have now been determined at resolutions of 1.95 and 1.85 {angstrom}, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformationmore » and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH{sub 3}-H{sub 4}folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops {beta}2-{alpha}2 (L2), {beta}3-{alpha}3 (L3), and {beta}4-{alpha}4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a 'closed' conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an 'open' conformation to allow NADH to bind.« less

When the Ties That Bind Break.

ERIC Educational Resources Information Center

Sorohan, Erica Gordon

1994-01-01

Despite decades of management fads, U.S. businesses have practiced chain-of-command management. Only through changes in leadership can corporate America repair the damage to employee morale that stems as much from the way companies execute layoffs as from the layoffs themselves. (Author/JOW)

Structure of Full-length Drosophila Cryptochrome

PubMed Central

Zoltowski, Brian D.; Vaidya, Anand T.; Top, Deniz; Widom, Joanne; Young, Michael W.; Crane, Brian R.

2011-01-01

The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions. PMID:22080955

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huerta, Carlos; Borek, Dominika; Machius, Mischa

2009-12-01

Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme flavin adenine dinucleotide (FAD) and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different sets of active-site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from the pathogenic yeast Candida glabrata. Four crystal structures of C. glabrata FMNAT in different complexed forms were determined at 1.20-1.95 A resolutions, capturing the enzyme active-site states prior to and after catalysis. These structures reveal a novelmore » flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNATs provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. Structure-based investigation of the kinetic properties of FMNAT should offer insights into the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms.« less

Structural Basis for Substrate Fatty Acyl Chain Specificity

PubMed Central

McAndrew, Ryan P.; Wang, Yudong; Mohsen, Al-Walid; He, Miao; Vockley, Jerry; Kim, Jung-Ja P.

2008-01-01

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-Å resolution. The overall fold of the N-terminal ∼400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an α-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12Å and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial β-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype. PMID:18227065

Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (Haliotis discus discus): Insights as a potent player in host antioxidant defense.

PubMed

Herath, H M L P B; Wickramasinghe, P D S U; Bathige, S D N K; Jayasooriya, R G P T; Kim, Gi-Young; Park, Myoung Ae; Kim, Chul; Lee, Jehee

2017-01-01

Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H 2 O 2 , the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H 2 O 2 . Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171-246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu +2 and Cd +2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel L-amino acid oxidase from Trichoderma harzianum ETS 323 associated with antagonism of Rhizoctonia solani.

PubMed

Yang, Chia-Ann; Cheng, Chi-Hua; Lo, Chaur-Tsuen; Liu, Shu-Ying; Lee, Jeng-Woei; Peng, Kou-Cheng

2011-05-11

Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.

Label-free Raman spectroscopy for accessing intracellular anticancer drug release on gold nanoparticles.

PubMed

Ock, Kwang-Su; Ganbold, Erdene Ochir; Park, Jin; Cho, Keunchang; Joo, Sang-Woo; Lee, So Yeong

2012-06-21

We investigated glutathione (GSH)-induced purine or pyrimidine anticancer drug release on gold nanoparticle (AuNP) surfaces by means of label-free Raman spectroscopy. GSH-triggered releases of 6-thioguanine (6TG), gemcitabine (GEM), acycloguanosine (ACY), and fadrozole (FAD) were examined in a comparative way by means of surface-enhanced Raman scattering (SERS). The GSH-induced dissociation constant of GEM (or ACY/FAD) from AuNPs was estimated to be larger by more than 38 times than that of 6TG from the kinetic relationship. Tripeptide control experiments were presented to check the turn-off Raman signalling mechanism. Dark-field microscopy (DFM) and transmission electron microscopy (TEM) indicated the intracellular AuNP loads. After their cellular uptake, GEM, ACY, and FAD would not show SERS intensities as strong as 6TG. This may be due to easier release of GEM, ACY, and FAD than 6TG by intracellular reducing species including GSH. We observed fairly strong SERS signals of GEM and 6TG in cell culture media solution. Our CCK-8 cytotoxicity assay data support that 6TG-AuNPs did not exhibit a substantial decrease in cell viability presumably due to strong binding. Label-free confocal Raman spectroscopy can be utilized as an effective tool to access intracellular anticancer drug release.

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.

PubMed

Iwata, Momi; Lee, Yang; Yamashita, Tetsuo; Yagi, Takao; Iwata, So; Cameron, Alexander D; Maher, Megan J

2012-09-18

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.

Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.

PubMed Central

Marin, E; Nussaume, L; Quesada, A; Gonneau, M; Sotta, B; Hugueney, P; Frey, A; Marion-Poll, A

1996-01-01

Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location. Images PMID:8665840

Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.

PubMed

Marin, E; Nussaume, L; Quesada, A; Gonneau, M; Sotta, B; Hugueney, P; Frey, A; Marion-Poll, A

1996-05-15

Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location.

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.

PubMed

Kim, Chang-Yub; Webster, Cecelia; Roberts, Justin K M; Moon, Jin Ho; Alipio Lyon, Emily Z; Kim, Heungbok; Yu, Minmin; Hung, Li-Wei; Terwilliger, Thomas C

2009-12-01

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

PubMed Central

Zhang, Xinxing; Jones, Rachel A.; Bruner, Steven D.; Butcher, Rebecca A.

2016-01-01

Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state. PMID:27551084

Structural basis of kynurenine 3-monooxygenase inhibition.

PubMed

Amaral, Marta; Levy, Colin; Heyes, Derren J; Lafite, Pierre; Outeiro, Tiago F; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S

2013-04-18

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer's disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases.

Outer-membrane Transport of Aromatic Hydrocarbons as a First Step in Biodegradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hearn,E.; Patel, D.; van den Berg, B.

Bacterial biodegradation of hydrocarbons, an important process for environmental remediation, requires the passage of hydrophobic substrates across the cell membrane. Here, we report crystal structures of two outer membrane proteins, Pseudomonas putida TodX and Ralstonia pickettii TbuX, which have been implicated in hydrocarbon transport and are part of a subfamily of the FadL fatty acid transporter family. The structures of TodX and TbuX show significant differences with those previously determined for Escherichia coli FadL, which may provide an explanation for the substrate-specific transport of TodX and TbuX observed with in vivo transport assays. The TodX and TbuX structures revealed 14-strandedmore » {beta}-barrels with an N-terminal hatch domain blocking the barrel interior. A hydrophobic channel with bound detergent molecules extends from the extracellular surface and is contiguous with a passageway through the hatch domain, lined by both hydrophobic and polar or charged residues. The TodX and TbuX structures support a mechanism for transport of hydrophobic substrates from the extracellular environment to the periplasm via a channel through the hatch domain.« less

Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study.

PubMed

Sheng, Yinghong; Zhong, Linghao; Guo, Dahai; Lau, Gavin; Feng, Changjian

2015-12-01

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production. Copyright © 2015 Elsevier Inc. All rights reserved.

Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin

PubMed Central

Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

2017-01-01

Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation. PMID:28270505

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi

2013-06-01

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

A Cryptochrome 2 mutation yields advanced sleep phase in humans.

PubMed

Hirano, Arisa; Shi, Guangsen; Jones, Christopher R; Lipzen, Anna; Pennacchio, Len A; Xu, Ying; Hallows, William C; McMahon, Thomas; Yamazaki, Maya; Ptáček, Louis J; Fu, Ying-Hui

2016-08-16

Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.

Structural and Evolutionary Aspects of Antenna Chromophore Usage by Class II Photolyases*

PubMed Central

Kiontke, Stephan; Gnau, Petra; Haselsberger, Reinhard; Batschauer, Alfred; Essen, Lars-Oliver

2014-01-01

Light-harvesting and resonance energy transfer to the catalytic FAD cofactor are key roles for the antenna chromophores of light-driven DNA photolyases, which remove UV-induced DNA lesions. So far, five chemically diverse chromophores have been described for several photolyases and related cryptochromes, but no correlation between phylogeny and used antenna has been found. Despite a common protein topology, structural analysis of the distantly related class II photolyase from the archaeon Methanosarcina mazei (MmCPDII) as well as plantal orthologues indicated several differences in terms of DNA and FAD binding and electron transfer pathways. For MmCPDII we identify 8-hydroxydeazaflavin (8-HDF) as cognate antenna by in vitro and in vivo reconstitution, whereas the higher plant class II photolyase from Arabidopsis thaliana fails to bind any of the known chromophores. According to the 1.9 Å structure of the MmCPDII·8-HDF complex, its antenna binding site differs from other members of the photolyase-cryptochrome superfamily by an antenna loop that changes its conformation by 12 Å upon 8-HDF binding. Additionally, so-called N- and C-motifs contribute as conserved elements to the binding of deprotonated 8-HDF and allow predicting 8-HDF binding for most of the class II photolyases in the whole phylome. The 8-HDF antenna is used throughout the viridiplantae ranging from green microalgae to bryophyta and pteridophyta, i.e. mosses and ferns, but interestingly not in higher plants. Overall, we suggest that 8-hydroxydeazaflavin is a crucial factor for the survival of most higher eukaryotes which depend on class II photolyases to struggle with the genotoxic effects of solar UV exposure. PMID:24849603

Deciphering structural and functional roles of individual disulfide bonds of the mitochondrial sulfhydryl oxidase Erv1p.

PubMed

Ang, Swee Kim; Lu, Hui

2009-10-16

Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX(16)C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX(16)C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p*

PubMed Central

Ang, Swee Kim; Lu, Hui

2009-01-01

Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX16C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur. PMID:19679655

The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

PubMed Central

Dreveny, Ingrid; Kratky, Christoph; Gruber, Karl

2002-01-01

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction—the enantiospecific formation of α-hydroxynitriles—is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C–C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident. PMID:11790839

A New F131V Mutation in Chlamydomonas Phytoene Desaturase Locates a Cluster of Norflurazon Resistance Mutations near the FAD-Binding Site in 3D Protein Models

PubMed Central

Suarez, Julio V.; Banks, Stephen; Thomas, Paul G.; Day, Anil

2014-01-01

The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM). The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505) but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene. PMID:24936791

Structural basis of kynurenine 3-monooxygenase inhibition

PubMed Central

Amaral, Marta; Levy, Colin; Heyes, Derren J.; Lafite, Pierre; Outeiro, Tiago F.; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S.

2013-01-01

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (i.e. kynurenine pathway), leads to amelioration of Huntington’s disease-relevant phenotypes in yeast, fruit fly, and mouse models1–5, as well as a mouse model of Alzheimer’s disease3. KMO is a FAD-dependent monooxygenase, and is located in the outer mitochondrial membrane where it converts L-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders6, as well as cancer7,8, and several peripheral inflammatory conditions9. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained hitherto unknown. Here we report the first crystal structure of KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active site structure, preventing productive binding of the substrate kynurenine. Functional assays and targeted mutagenesis revealed that the active site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO:UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington’s, Alzheimer’s, and Parkinson’s diseases. PMID:23575632

The Alzheimer Pandemic: Is Paracetamol to Blame?

PubMed Central

Jones, Günther Robert Norman

2013-01-01

Historical Background: The clinical recognition of a form of dementia closely resembling Alzheimer's disease dates from around 1800. The role of analgesics derived from coal-tar in the spread of the pandemic is traced in terms of the introduction of phenacetin (PN) in 1887; its nephrotoxicity; the observation of lesions characteristic of the disease by Fischer and Alzheimer; the discovery of paracetamol (PA) as the major metabolite of PN; the linking of kidney injury and dementia with high PN usage; and the failure of PN replacement by PA to halt and reverse the exponential, inexorable rise in the incidence of Alzheimer-type dementia. Fischer observed his first case before Alzheimer; it is proposed to rename the syndrome Fischer-Alzheimer disease (F-AD). Disease development: PA-metabolising enzymes are localised in the synaptic areas of the frontal cortex and hippocampus, where F-AD lesions arise. The initiating chemical lesions in liver poisoning comprise covalent binding of a highly reactive product of PA metabolism to proteins; similar events are believed to occur in brain, where alterations in the antigenic profiles of cerebral proteins activate the microglia. β-Amyloid forms, and, like PA itself, induces nitric oxide synthase. Peroxynitrite modifies cerebral proteins by nitrating tyrosine residues, further challenging the microglia and exacerbating the amyloid cascade. Spontaneous reinnervation, N-acetyl cysteine administration and tyrosine supplementation may attenuate the early stages of F-AD development. Conclusion: F-AD is primarily a man-made condition with PA as its principal risk factor. PMID:24350947

Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm.

PubMed

Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V

2016-02-01

We recently reported a flavin-trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal-dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal-dependent Ftp catalysis in either Nqr- or Rnf-redox-containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD-binding protein to a Mg(2+)-dependent FAD pyrophosphatase (Ftp_Tp-like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

PubMed

Thambugala, Dinushika; Cloutier, Sylvie

2014-11-01

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Two ω-3 FADs Are Associated with Peach Fruit Volatile Formation

PubMed Central

Wang, Jiao-Jiao; Liu, Hong-Ru; Gao, Jie; Huang, Yu-Ji; Zhang, Bo; Chen, Kun-Song

2016-01-01

Aroma-related volatiles, together with sugars and acids, play an important role in determining fruit flavor quality. Characteristic volatiles of peach fruit are mainly derived from fatty acids such as linoleic acid (18:2) and linolenic acid (18:3). In the present study, six genes encoding fatty acid desaturases (FAD) were cloned, including two ω-6 FAD genes (PpFAD2, PpFAD6) and four ω-3 FAD genes (PpFAD3-1, PpFAD3-2, PpFAD7 and PpFAD8). Heterologous expression of peach FADs in tobacco plants showed that PpFAD3-1, and PpFAD3-2 significantly reduced contents of 18:2, and accumulated significant higher levels of 18:3. In the case of volatiles, transgenic plants produced lower concentrations of hexanal and higher levels of (E)-2-hexenal. Consequently, the ratio of the (E)-2-hexenal and hexanal was about 5- and 3-fold higher than that of wild type (WT) in PpFAD3-1 and PpFAD3-2 transformants, respectively. No significant changes in volatile profiles were observed in transgenic plants overexpressing the four other peach FAD genes. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that ripe fruit had high PpFAD3-1 and low PpFAD3-2 transcript levels. In contrast, high PpFAD3-2 and low PpFAD3-1 transcript levels were observed in young fruit. These results indicate a temporal regulation of these two ω-3 FADs during development and ripening, influencing peach fruit volatile formation. PMID:27043529

Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

PubMed

Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

2010-03-23

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Shwing a Covalent Flavin-Aspartate Bond

DOE Office of Scientific and Technical Information (OSTI.GOV)

Podzelinska, K.; Latimer, R; Bhattacharya, A

2010-01-01

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 {angstrom} resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, themore » C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4{alpha})-OOH intermediate. Strikingly, the 8{alpha} carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.« less

Effects of Addition of Linseed and Marine Algae to the Diet on Adipose Tissue Development, Fatty Acid Profile, Lipogenic Gene Expression, and Meat Quality in Lambs

PubMed Central

Urrutia, Olaia; Mendizabal, José Antonio; Insausti, Kizkitza; Soret, Beatriz; Purroy, Antonio; Arana, Ana

2016-01-01

This study examined the effect of linseed and algae on growth and carcass parameters, adipocyte cellularity, fatty acid profile and meat quality and gene expression in subcutaneous and intramuscular adipose tissues (AT) in lambs. After weaning, 33 lambs were fed three diets up to 26.7 ± 0.3 kg: Control diet (barley and soybean); L diet (barley, soybean and 10% linseed) and L-A diet (barley, soybean, 5% linseed and 3.89% algae). Lambs fed L-A diet showed lower average daily gain and greater slaughter age compared to Control and L (P < 0.001). Carcass traits were not affected by L and L-A diets, but a trend towards greater adipocyte diameter was observed in L and L-A in the subcutaneous AT (P = 0.057). Adding either linseed or linseed and algae increased α-linolenic acid and eicosapentaenoic acid contents in both AT (P < 0.001); however, docosahexaenoic acid was increased by L-A (P < 0.001). The n-6/n-3 ratio decreased in L and L-A (P < 0.001). Algae had adverse effects on meat quality, with greater lipid oxidation and reduced ratings for odor and flavor. The expression of lipogenic genes was downregulated in the subcutaneous AT (P < 0.05): acetyl-CoA carboxylase 1 (ACACA) in L and L-A and lipoprotein lipase (LPL) and stearoyl-CoA desaturase (SCD) in L-A. Fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2) and fatty acid elongase 5 (ELOVL5) were unaffected. In the subcutaneous AT, supplementing either L or L-A increased peroxisome proliferator-activated receptor gamma (PPARG) and CAAT-enhancer binding protein alpha (CEBPA) (P < 0.05), although it had no effect on sterol regulatory element-binding factor 1 (SREBF1). In the intramuscular AT, expression of ACACA, SCD, FADS1 and FADS2 decreased in L and L-A (P < 0.001) and LPL in L (P < 0.01), but PPARG, CEBPA and SREBF1 were unaffected. PMID:27253325

Tissue-specific impact of FADS cluster variants on FADS1 and FADS2 gene expression.

PubMed

Reynolds, Lindsay M; Howard, Timothy D; Ruczinski, Ingo; Kanchan, Kanika; Seeds, Michael C; Mathias, Rasika A; Chilton, Floyd H

2018-01-01

Omega-6 (n-6) and omega-3 (n-3) long (≥ 20 carbon) chain polyunsaturated fatty acids (LC-PUFAs) play a critical role in human health and disease. Biosynthesis of LC-PUFAs from dietary 18 carbon PUFAs in tissues such as the liver is highly associated with genetic variation within the fatty acid desaturase (FADS) gene cluster, containing FADS1 and FADS2 that encode the rate-limiting desaturation enzymes in the LC-PUFA biosynthesis pathway. However, the molecular mechanisms by which FADS genetic variants affect LC-PUFA biosynthesis, and in which tissues, are unclear. The current study examined associations between common single nucleotide polymorphisms (SNPs) within the FADS gene cluster and FADS1 and FADS2 gene expression in 44 different human tissues (sample sizes ranging 70-361) from the Genotype-Tissue Expression (GTEx) Project. FADS1 and FADS2 expression were detected in all 44 tissues. Significant cis-eQTLs (within 1 megabase of each gene, False Discovery Rate, FDR<0.05, as defined by GTEx) were identified in 12 tissues for FADS1 gene expression and 23 tissues for FADS2 gene expression. Six tissues had significant (FDR< 0.05) eQTLs associated with both FADS1 and FADS2 (including artery, esophagus, heart, muscle, nerve, and thyroid). Interestingly, the identified eQTLs were consistently found to be associated in opposite directions for FADS1 and FADS2 expression. Taken together, findings from this study suggest common SNPs within the FADS gene cluster impact the transcription of FADS1 and FADS2 in numerous tissues and raise important questions about how the inverse expression of these two genes impact intermediate molecular (such a LC-PUFA and LC-PUFA-containing glycerolipid levels) and ultimately clinical phenotypes associated with inflammatory diseases and brain health.

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase.

PubMed

Ouedraogo, Daniel; Souffrant, Michael; Vasquez, Sheena; Hamelberg, Donald; Gadda, Giovanni

2017-05-16

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k cat /K m values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k cat value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

Decomposition of the fluorescence spectra of two FAD molecules in electron-transferring flavoprotein from Megasphaera elsdenii.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi

2013-07-01

Electron-transferring flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules, FAD-1 and FAD-2. FAD-2 shows an unusual absorption spectrum with a 400-nm peak. In contrast, ETFs from other sources such as pig contain one FAD and one AMP with the FAD showing a typical flavin absorption spectrum with 380- and 440-nm peaks. It is presumed that FAD-2 is the counterpart of the FAD in other ETFs. In this study, the FAD-1 and FAD-2 fluorescence spectra were determined by titration of FAD-1-bound ETF with FAD using excitation-emission matrix (EEM) fluorescence spectroscopy. The EEM data were globally analysed, and the FAD fluorescence spectra were calculated from the principal components using their respective absorption spectra. The FAD-2 fluorescence spectrum was different from that of pig ETF, which is more intense and blue-shifted. AMP-free pig ETF in acidic solution, which has a comparable absorption spectrum to FAD-2, also had a similar fluorescence spectrum. This result suggests that FAD-2 in M. elsdenii ETF and the FAD in acidic AMP-free pig ETF share a common microenvironment. A review of published ETF fluorescence spectra led to the speculation that the majority of ETF molecules in solution are in the conformation depicted by the crystal structure.

Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

PubMed Central

Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

2015-01-01

Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.

PubMed

Subrahmanyam, S; Cronan, J E

1999-01-21

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.

Lactate Utilization Is Regulated by the FadR-Type Regulator LldR in Pseudomonas aeruginosa

PubMed Central

Gao, Chao; Hu, Chunhui; Zheng, Zhaojuan; Jiang, Tianyi; Dou, Peipei; Zhang, Wen; Che, Bin; Wang, Yujiao; Lv, Min

2012-01-01

NAD-independent l-lactate dehydrogenase (l-iLDH) and NAD-independent d-lactate dehydrogenase (d-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an l-iLDH), and lldE (encoding a d-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses d-iLDH and l-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldRM, lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. l-Lactate and d-lactate interfered with the DNA-binding activity of LldR. Thus, l-iLDH and d-iLDH were expressed when the operon was induced in the presence of l-lactate or d-lactate. PMID:22408166

The GmFAD7 gene family from soybean: identification of novel genes and tissue-specific conformations of the FAD7 enzyme involved in desaturase activity.

PubMed

Andreu, Vanesa; Lagunas, Beatriz; Collados, Raquel; Picorel, Rafael; Alfonso, Miguel

2010-07-01

The FAD7 gene encodes a omega3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean omega3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular omega3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity.

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea.

PubMed

Lee, Kyeong-Ryeol; In Sohn, Soo; Jung, Jin Hee; Kim, Sun Hee; Roh, Kyung Hee; Kim, Jong-Bum; Suh, Mi Chung; Kim, Hyun Uk

2013-12-01

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns. © 2013.

Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

PubMed Central

Morrison, Eliot; Kantz, Auric; Gassner, George T.; Sazinsky, Matthew H.

2013-01-01

The two–component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH– and FAD–dependent epoxidation of styrene to styrene oxide. In this study we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to epoxidase (NSMOA) component and report our findings in light of the 2.2–Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH→FADox charge–transfer intermediate and catalyzes a hydride–transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s−1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium–binding studies indicate that NSMOA binds FADhq ~13–times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)–hydroperoxide intermediate. The half–life of apoSMOB generated in the FAD–transfer reaction is increased ~21–fold, supporting the model of a protein–protein interaction between apoSMOB and NSMOA with the peroxide intermediate. The mechanisms of FAD–dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. Based on these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S–state) and more reactive, transfer state (T–state). Dissociation of NAD+ after the hydride transfer–reaction transiently populates the T–state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB–FADhq shifts the FADhq–binding equilibrium from the T–state to the S–state. Additionally, the 2.2–Å crystal structure of SMOB–FADox reported in this work is discussed in light of the pyridine nucleotide–gated flavin–transfer and electron–transfer reactions. PMID:23909369

Cellobiose dehydrogenase of Chaetomium sp. INBI 2-26(-): structural basis of enhanced activity toward glucose at neutral pH.

PubMed

Vasilchenko, Liliya G; Karapetyan, Karen N; Yershevich, Olga P; Ludwig, Roland; Zamocky, Marcel; Peterbauer, Clemens K; Haltrich, Dietmar; Rabinovich, Mikhail L

2011-05-01

Cellobiose dehydrogenase (CDH) is an extracellular fungal flavocytochrome specifically oxidizing cellooligosaccharides and lactose to corresponding (-lactones by a variety of electron acceptors. In contrast to basidiomycetous CDHs, CDHs of ascomycetes also display certain activity toward glucose. The objective of this study was to establish the structural reasons of such an activity of CDH from mesophilic ascomycete Chaetomium sp. INBI 2-26 (ChCDH). The complete amino acid sequence of ChCDH displayed high levels of similarity with the amino acid sequences of CDHs from the thermophilic fungi Thielavia heterotallica and Myriococcum thermophilum. Peptide mass fingerprinting of purified ChCDH provided evidence for the oxidation of methionine residues in the FAD-domain. Comparative homology modeling of the structure of the ChCDH FAD-domain in complex with the transition state analog based on the structure of the same complex of basidiomycetous CDH (1NAA) as template indicated possible structural reasons for the enhanced activity of ascomycetous CDHs toward glucose at neutral pH, which is a prerequisite for application of CDH in a variety of biocompatible biosensors and biofuel cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DEER distance measurement between a spin label and a native FAD semiquinone in electron transfer flavoprotein.

PubMed

Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

2009-11-11

The human mitochondrial electron transfer flavoprotein (ETF) accepts electrons from at least 10 different flavoprotein dehydrogenases and transfers electrons to a single electron acceptor in the inner membrane. Paracoccus denitrificans ETF has the identical function, shares the same three-dimensional structure and functional domains, and exhibits the same conformational mobility. It has been proposed that the mobility of the alphaII domain permits the promiscuous behavior of ETF with respect to a variety of redox partners. Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in P. denitrificans ETF gave two distributions of distances: a major component centered at 4.2 +/- 0.1 nm and a minor component centered at 5.1 +/- 0.2 nm. Both components had widths of approximately 0.3 nm. A distance of 4.1 nm was calculated using the crystal structure of P. denitrificans ETF, which agrees with the major component obtained from the DEER measurement. The observation of a second distribution suggests that ETF, in the absence of substrate, adopts some conformations that are intermediate between the predominant free and substrate-bound states.

DEER Distance Measurement Between a Spin Label and a Native FAD Semiquinone in Electron Transfer Flavoprotein

PubMed Central

Swanson, Michael A.; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

2009-01-01

The human mitochondrial electron transfer flavoprotein (ETF) accepts electrons from at least 10 different flavoprotein dehydrogenases and transfers electrons to a single electron acceptor in the inner membrane. Paracoccus denitrificans ETF has the identical function, shares the same three dimensional structure and functional domains, and exhibits the same conformational mobility. It has been proposed that the mobility of the αII domain permits the promiscuous behavior of ETF with respect to a variety of redox partners. Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in P. denitrificans ETF gave two distributions of distances: a major component centered at 4.2 ± 0.1 nm and a minor component centered at 5.1 ± 0.2 nm. Both components had widths of approximately 0.3 nm. A distance of 4.1 nm was calculated using the crystal structure of P. denitrificans ETF, which agrees with the major component obtained from the DEER measurement. The observation of a second distribution suggests that ETF, in the absence of substrate, adopts some conformations that are intermediate between the predominant free and substrate-bound states. PMID:19886689

Either fadD1 or fadD2, Which Encode acyl-CoA Synthetase, Is Essential for the Survival of Haemophilus parasuis SC096.

PubMed

Feng, Saixiang; Xu, Chenggang; Yang, Kaijie; Wang, Haihong; Fan, Huiying; Liao, Ming

2017-01-01

In Haemophilus parasuis , the genes HAPS_0217 and HAPS_1695 are predicted to encode long-chain fatty acid-CoA ligases (FACSs). These proteins contain ATP/AMP signature motifs and FACS conserved motifs that are homologous to those in Escherichia coli FadD (EcFadD). In this study, we demonstrate that HAPS_0217 and HAPS_1695 can functionally replace EcFadD in the E. coli fadD mutant JW1794, and were thus designated fadD1 and fadD2 , respectively. An evaluation of kinetic parameters indicated that FadD1 and FadD2 have a substrate preference for long-chain fatty acids. Moreover, FadD2 exhibited substrate inhibition in the presence of high concentrations of oleic acid. Single mutants of each of the fadD genes were easily constructed, whereas double mutants were not. These results were further confirmed using genomic site-directed mutagenesis, which supported the idea that H. parasuis requires either fadD1 or fadD2 for survival. The fadD1 mutant exhibited slower growth than the wild-type strain SC096, and its complementation resulted in a restored phenotype. The wild-type strain did not grow on chemically defined medium without the addition of oleic acid, indicating that lipids are a vital nutrient for this bacterium. Additionally, strains with a disrupted fadD1 gene also exhibited increased sensitivity to quinolone antibiotics, including levofloxacin, enrofloxacin, ciprofloxacin and nalidixic acid.

Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

PubMed

Monroig, Oscar; Zheng, Xiaozhong; Morais, Sofia; Leaver, Michael J; Taggart, John B; Tocher, Douglas R

2010-09-01

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology. 2010 Elsevier B.V. All rights reserved.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

NASA Astrophysics Data System (ADS)

Jahn, Karolina; Buschmann, Volker; Hille, Carsten

2015-09-01

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase

PubMed Central

Gemmecker, Sandra; Poussin-Courmontagne, Pierre; Mailliot, Justine; McEwen, Alastair G.; Ghisla, Sandro; Al-Babili, Salim; Cavarelli, Jean; Beyer, Peter

2012-01-01

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C40 hydrocarbon substrate. PMID:22745782

A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

PubMed

Bifulco, Davide; Pollegioni, Loredano; Tessaro, Davide; Servi, Stefano; Molla, Gianluca

2013-08-01

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

Broad spectrum protection of broiler chickens against inclusion body hepatitis by immunizing their broiler breeder parents with a bivalent live fowl adenovirus vaccine.

PubMed

Popowich, Shelly; Gupta, Ashish; Chow-Lockerbie, Betty; Ayalew, Lisanework; Ambrose, Neil; Ojkic, Davor; Gunawardana, Thushari; Kurukulasuriya, Shanika; Willson, Philip; Tikoo, Suresh K; Gomis, Susantha

2018-06-01

Historically, fowl adenovirus (FAdV) associated inclusion body hepatitis (IBH) was considered a secondary disease in broiler chickens associated with immunosuppression. However, we previously reported the occurrence of IBH as a primary disease in the broiler chicken industry in Canada as a result of infections with various FAdV serotypes. Therefore, the objectives of this study were to develop an immunization strategy in broiler breeders using live FAdV 11-1047 and FAdV8a-TR59 to confer homologous and heterologous protection in broiler progeny against IBH and to study the efficacy of natural exposure of naïve broiler breeders to a vaccine virus from live FAdV vaccinated birds as an immunization technique. Broiler breeders vaccinated orally with FAdV8a-TR59 (1 × 10 4 TCID 50 /bird) and FAdV11-1047 (1 × 10 4 TCID 50 /bird), FAdV8a-TR59 (1 × 10 6 TCID 50 /bird) and FAdV11-1047 (1 × 10 6 TCID 50 /bird) or FAdV8b (1 × 10 6 TCID 50 /bird) transferred substantial levels of neutralizing antibodies to their progeny. The efficacy of maternal antibodies was studied by challenging 14-day old broiler chickens with 1 × 10 7 TCID 50 of FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 which are the dominant serotypes causing IBH outbreaks in Canada. Broiler chickens from the low and high dose vaccinated breeders were significantly protected against all serotypes of FAdV (P < 0.05). Comingling of unvaccinated broiler breeders with FAdV-vaccinated broiler breeders was an effective immunization technique for in-contact naïve birds. This study confirms that IBH can be effectively controlled in Canada by vaccination of broiler breeder parents with a bivalent vaccine containing live FAdV8a-TR59 and FAdV11-1047. Copyright © 2018 Elsevier Ltd. All rights reserved.

FAD oxidizes the ERO1-PDI electron transfer chain: The role of membrane integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papp, Eszter; Nardai, Gabor; Mandl, Jozsef

2005-12-16

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1more » can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.« less

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

PubMed

Demorest, Zachary L; Coffman, Andrew; Baltes, Nicholas J; Stoddard, Thomas J; Clasen, Benjamin M; Luo, Song; Retterath, Adam; Yabandith, Ann; Gamo, Maria Elena; Bissen, Jeff; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

2016-10-13

The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A (FAD2-1A) and FAD2-1B genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within FAD2-1A and FAD2-1B with mutations in fatty acid desaturase 3A (FAD3A) to further decrease levels of linolenic acid. Mutations were introduced into FAD3A by directly delivering TALENs into fad2-1a fad2-1b soybean plants. Oil from fad2-1a fad2-1b fad3a plants had significantly lower levels of linolenic acid (2.5 %), as compared to fad2-1a fad2-1b plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free fad2-1a fad2-1b fad3a soybean lines were identified. The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.

A single nucleotide polymorphism in the FADS1/FADS2 gene is associated with plasma lipid profiles in two genetically similar Asian ethnic groups with distinctive differences in lifestyle.

PubMed

Nakayama, Kazuhiro; Bayasgalan, Tumenbayer; Tazoe, Fumiko; Yanagisawa, Yoshiko; Gotoh, Takaya; Yamanaka, Kazuhiro; Ogawa, Ayumi; Munkhtulga, Lkhagvasuren; Chimedregze, Ulziiburen; Kagawa, Yasuo; Ishibashi, Shun; Iwamoto, Sadahiko

2010-06-01

Recent genome-wide association studies (GWASs) showed that single nucleotide polymorphisms (SNPs) in FADS1/FADS2 were associated with plasma lipid concentrations in populations with European ancestry. We investigated the associations between the SNPs in FADS1/FADS2 and plasma concentrations of triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in two Asian groups, i.e., Japanese and Mongolians. The genotype of rs174547 (T/C), found to be associated with triglyceride and HDL-C concentrations in the GWAS, was determined in 21,004 Japanese and 1,203 Mongolian individuals. Genotype-phenotype association was assessed by using multiple linear regression models, assuming an additive model of inheritance. The copy number of the rs174547 C allele was significantly associated with increased triglyceride levels (P = 1.5 x 10(-6)) and decreased HDL-C levels (P = 0.03) in the Japanese population. On the other hand, in the Mongolian population, the rs174547 C allele copy number was strongly associated with decreased LDL-C levels (P = 2.6 x 10(-6)), but was not associated with triglyceride and HDL-C levels. The linkage disequilibrium pattern and haplotype structures of SNPs around the FADS1/FADS2 locus showed no marked dissimilarity between Japanese and Mongolian individuals. The present data indicate that the FADS1/FADS2 locus can be added to the growing list of loci involved in polygenic dyslipidemia in Asians. Furthermore, the variable effects of FADS1/FADS2 on plasma lipid profiles in Asians may result from differences in the dietary intake of polyunsaturated fatty acids, which serve as substrates for enzymes encoded by FADS1/FADS2.

Fads1 and 2 are promoted to meet instant need for long-chain polyunsaturated fatty acids in goose fatty liver.

PubMed

Osman, Rashid H; Liu, Long; Xia, Lili; Zhao, Xing; Wang, Qianqian; Sun, Xiaoxian; Zhang, Yihui; Yang, Biao; Zheng, Yun; Gong, Daoqing; Geng, Tuoyu

2016-07-01

Global prevalence of non-alcoholic fatty liver disease (NAFLD) constitutes a threat to human health. Goose is a unique model of NAFLD for discovering therapeutic targets as its liver can develop severe steatosis without overt injury. Fatty acid desaturase (Fads) is a potential therapeutic target as Fads expression and mutations are associated with liver fat. Here, we hypothesized that Fads was promoted to provide a protection for goose fatty liver. To test this, goose Fads1 and Fads2 were sequenced. Fads1/2/6 expression was determined in goose liver and primary hepatocytes by quantitative PCR. Liver fatty acid composition was also analyzed by gas chromatography. Data indicated that hepatic Fads1/2/6 expression was gradually increased with the time of overfeeding. In contrast, trans-C18:1n9 fatty acid (Fads inhibitor) was reduced. However, enhanced Fads capacity for long-chain polyunsaturated fatty acid (LC-PUFA) synthesis was not sufficient to compensate for the depleted LC-PUFAs in goose fatty liver. Moreover, cell studies showed that Fads1/2/6 expression was regulated by fatty liver-associated factors. Together, these findings suggest Fads1/2 as protective components are promoted to meet instant need for LC-PUFAs in goose fatty liver, and we propose this is required for severe hepatic steatosis without liver injury.

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

PubMed

Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

2011-06-01

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns of fad3a and fad3b were highly correlated with linolenic acid accumulation during seed development, with the exception of fad3b in SP2047 whose lack of activity was caused by the histidine box mutation despite its transcript accumulation being similar to that of the fad3b of the other genotypes.

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

PubMed Central

Haigler, B E; Suen, W C; Spain, J C

1996-01-01

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases. PMID:8830701

The contribution of nearshore fish aggregating devices (FADs) to food security and livelihoods in Solomon Islands.

PubMed

Albert, Joelle A; Beare, Doug; Schwarz, Anne-Maree; Albert, Simon; Warren, Regon; Teri, James; Siota, Faye; Andrew, Neil L

2014-01-01

Fish aggregating devices, or FADs, are used widely in developing countries to concentrate pelagic fish, making them easier to catch. Nearshore FADs anchored close to the coast allow access for rural communities, but despite their popularity among policy makers, there is a dearth of empirical analysis of their contributions to the supply of fish and to fisheries management. In this paper we demonstrate that nearshore FADs increased the supply of fish to four communities in Solomon Islands. Estimated total annual fish catch ranged from 4300 to 12,000 kg across the study villages, with nearshore FADs contributing up to 45% of the catch. While it is clear that FADs increased the supply of fish, FAD catch rates were not consistently higher than other fishing grounds. Villages with limited access to diverse or productive fishing grounds seemingly utilized FADs to better effect. Villagers believed FADs increased household income and nutrition, as well as providing a source of fish for community events. FADs were also perceived to increase intra-household conflict and reduce fishers' participation in community activities. FADs need to be placed within a broader rural development context and treated as another component in the diversified livelihoods of rural people; as with other livelihood options they bring trade-offs and risks.

Absorption and fluorescence spectroscopic characterisation of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry)

NASA Astrophysics Data System (ADS)

Shirdel, J.; Zirak, P.; Penzkofer, A.; Breitkreuz, H.; Wolf, E.

2008-09-01

The absorption and fluorescence behaviour of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry) in a pH 8 aqueous buffer solution is studied. The flavin adenine dinucleotide (FAD) cofactor of dCry is identified to be present in its oxidized form (FAD ox), and the 5,10-methenyltetrahydrofolate (MTHF) cofactor is found to be hydrolyzed and oxidized to 10-formyldihydrofolate (10-FDHF). The absorption and the fluorescence behaviour of dCry is investigated in the dark-adapted (receptor) state, the light-adapted (signalling) state, and under long-time violet light exposure. Photo-excitation of FAD ox in dCry causes a reductive electron transfer to the formation of anionic FAD semiquinone (FAD rad - ), and photo-excitation of the generated FAD rad - causes an oxidative electron transfer to the back formation of FAD ox. In light adapted dCry a photo-induced equilibrium between FAD ox and FAD rad - exists. The photo-cycle dynamics of signalling state formation and recovery is discussed. Quantum yields of photo-induced signalling state formation of about 0.2 and of photo-induced back-conversion of about 0.2 are determined. A recovery of FAD rad - to FAD ox in the dark with a time constant of 1.6 min at room temperature is found.

The Contribution of Nearshore Fish Aggregating Devices (FADs) to Food Security and Livelihoods in Solomon Islands

PubMed Central

Albert, Joelle A.; Beare, Doug; Schwarz, Anne-Maree; Albert, Simon; Warren, Regon; Teri, James; Siota, Faye; Andrew, Neil L.

2014-01-01

Fish aggregating devices, or FADs, are used widely in developing countries to concentrate pelagic fish, making them easier to catch. Nearshore FADs anchored close to the coast allow access for rural communities, but despite their popularity among policy makers, there is a dearth of empirical analysis of their contributions to the supply of fish and to fisheries management. In this paper we demonstrate that nearshore FADs increased the supply of fish to four communities in Solomon Islands. Estimated total annual fish catch ranged from 4300 to 12 000 kg across the study villages, with nearshore FADs contributing up to 45% of the catch. While it is clear that FADs increased the supply of fish, FAD catch rates were not consistently higher than other fishing grounds. Villages with limited access to diverse or productive fishing grounds seemingly utilized FADs to better effect. Villagers believed FADs increased household income and nutrition, as well as providing a source of fish for community events. FADs were also perceived to increase intra-household conflict and reduce fishers' participation in community activities. FADs need to be placed within a broader rural development context and treated as another component in the diversified livelihoods of rural people; as with other livelihood options they bring trade-offs and risks. PMID:25513808

Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily

PubMed Central

Kim, Eun-Mi; Seo, Joo-Hyun; Baek, Kiheon; Kim, Byung-Gee

2015-01-01

Herein, we report a two-step deglycosylation mediated by the oxidation of glycoside which is different from traditional glycoside hydrolase (GH) mechanism. Previously, we reported a novel flavin adenine dinucleotide (FAD)-dependent glycoside oxidoreductase (FAD-GO) having deglycosylation activity. Various features of the reaction of FAD-GO such as including mechanism and catalytic residue and substrate specificity were studied. In addition, classification of novel FAD-GO subfamily was attempted. Deglycosylation of glycoside was performed spontaneously via oxidation of 3-OH of glycone moiety by FAD-GO mediated oxidation reaction. His493 residue was identified as a catalytic residue for the oxidation step. Interestingly, this enzyme has broad glycone and aglycon specificities. For the classification of FAD-GO enzyme subfamily, putative FAD-GOs were screened based on the FAD-GO from Rhizobium sp. GIN611 (gi 365822256) using BLAST search. The homologs of R. sp. GIN611 included the putative FAD-GOs from Stenotrophomonas strains, Sphingobacterium strains, Agrobacterium tumefaciens str. C58, and etc. All the cloned FAD-GOs from the three strains catalyzed the deglycosylation via enzymatic oxidation. Based on their substrate specificities, deglycosylation and oxidation activities to various ginsenosides, the FAD-GO subfamily members can be utilized as novel biocatalysts for the production of various aglycones. PMID:26057169

Application of high-resolution melting curve analysis for typing of fowl adenoviruses in field cases of inclusion body hepatitis.

PubMed

Steer, P A; O'Rourke, D; Ghorashi, S A; Noormohammadi, A H

2011-05-01

Fowl adenoviruses (FAdVs) cause inclusion body hepatitis (IBH) in chickens. In this study, clinical cases of IBH from Australian broiler flocks were screened for the presence and genotype of FAdVs. Twenty-six IBH cases from commercial poultry farms were screened. Polymerase chain reaction (PCR) coupled with high-resolution melt (HRM) curve analysis (PCR/HRM genotyping) was used to determine the presence and genotype of FAdVs. For comparison, field isolates were also assessed by virus microneutralisation and nucleotide sequence analysis of the hexon loop 1 (Hex L1) gene. PCR detection of chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) was also employed. FAdV-8b and FAdV-11 were identified in 13 cases each. In one case, FAdV-1 was also identified. Cross-neutralisation was observed between the FAdV-11 field strain and the reference FAdV-2 and 11 antisera, a result also seen with the type 2 and 11 reference FAdVs. Field strains 1 and 8b were neutralised only by their respective type antisera. The FAdV-8b field strain was identical to the Australian FAdV vaccine strain (type 8b) in the Hex L1 region. The Hex L1 sequence of the FAdV-11 field strain had the highest identity to FAdV-11 (93.2%) and FAdV-2 (92.7%) reference strains. In the five cases tested for CAV and IBDV, neither virus was detected. The evidence suggested the presence of sufficient antibodies against CAV and IBD in the parent flocks and there was no indication of immunosuppression caused by these viruses. These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species. © 2011 The Authors. Australian Veterinary Journal © 2011 Australian Veterinary Association.

Two Δ6-desaturase-like genes in common carp (Cyprinus carpio var. Jian): structure characterization, mRNA expression, temperature and nutritional regulation.

PubMed

Ren, Hong-tao; Zhang, Guang-qin; Li, Jian-lin; Tang, Yong-kai; Li, Hong-xia; Yu, Ju-hua; Xu, Pao

2013-08-01

Δ6-Desaturase is the rate-limiting enzyme involved in highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Δ6Fad-a and Δ6Fad-b in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Δ6-desaturase-like genes in common carp genome were obtained. We investigated the mRNA expression patterns of Δ6Fad-a and Δ6Fad-b in tissue, hatching carp embryos, larvae by temperature shock and juveniles under nutritional regulation. Our results showed that the two Δ6Fad genes had identical coding exon structures, being comprised of 12 coding exons, and with introns of distinct size and sequence composition. They were not allelic variants of a single gene. Both Δ6Fad genes were highly expressed in liver, intestine (pyloric caeca) and brain. The Δ6Fad-a and Δ6Fad-b mRNAs showed an increase in expression from newly hatched to 25 days after hatching. The expression levels of Δ6Fad-a were obviously regulated by temperature, whereas Δ6Fad-b was not affected by temperature. The regulation of Δ6Fad-a and Δ6Fad-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: diet1with fish oil (FO) rich in n-3 HUFA, diet2 with corn oil (CO, 18:2n-6) and diet3 with linseed oil (LO, 18:3n-3). The differential expression of Δ6Fad-a and Δ6Fad-b genes in liver, brain and intestine in common carps was fed with different oil sources, respectively. Further work is in progress to determine the mechanism of differential expression of the Δ6Fad-a and Δ6Fad-b genes in different tissues and the roles of transcription factors in regulating HUFA synthesis. Copyright © 2013 Elsevier B.V. All rights reserved.

The family assessment device: an update.

PubMed

Mansfield, Abigail K; Keitner, Gabor I; Dealy, Jennifer

2015-03-01

The current study set out to describe family functioning scores of a contemporary community sample, using the Family Assessment Device (FAD), and to compare this to a currently help-seeking sample. The community sample consisted of 151 families who completed the FAD. The help-seeking sample consisted of 46 families who completed the FAD at their first family therapy appointment as part of their standard care at an outpatient family therapy clinic at an urban hospital. Findings suggest that FAD means from the contemporary community sample indicate satisfaction with family functioning, while FAD scores from the help-seeking sample indicate dissatisfaction with family functioning. In addition, the General Functioning scale of the FAD continues to correlate highly with all other FAD scales, except Behavior Control. The cut-off scores for the FAD indicating satisfaction or dissatisfaction by family members with their family functioning continue to be relevant and the FAD continues to be a useful tool to assess family functioning in both clinical and research contexts. © 2014 Family Process Institute.

Facebook Addiction Disorder (FAD) among German students-A longitudinal approach.

PubMed

Brailovskaia, Julia; Margraf, Jürgen

2017-01-01

The present study aimed to investigate Facebook Addiction Disorder (FAD) in a German student sample over a period of one year. While mean FAD level did not increase during the investigation period, a significant increase was shown in the number of participants reaching the critical cutoff score. FAD was significantly positively related to the personality trait narcissism and to negative mental health variables (depression, anxiety, and stress symptoms). Furthermore, FAD fully mediated the significant positive relationship between narcissism and stress symptoms, which demonstrates that narcissistic people can be specifically at risk to develop FAD. Present results give a first overview of FAD in Germany. Practical applications for future studies and limitations of present results are discussed.

Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness

PubMed Central

Matteucci, M.; D'Angeli, S.; Errico, S.; Lamanna, R.; Perrotta, G.; Altamura, M. M.

2011-01-01

The olive tree lacks dormancy and is low temperature sensitive, with differences in cold tolerance and oil quality among genotypes. The oil is produced in the drupe, and the unsaturated fatty acids contribute to its quality. The aim of the present research was to investigate the relationship among development, cold response, expression of fatty acid desaturase (FAD) genes, and unsaturated fatty acid composition in drupes belonging to genotypes differing in leaf cold tolerance, but producing good oil (i.e. the non-hardy Moraiolo, the semi-hardy Frantoio, and the hardy Canino). In all genotypes, cold sensitivity, evaluated by cold-induced transient increases in cytosolic calcium, was high in the epi-mesocarp cells before oil body formation, and decreased during oil biogenesis. However, genotype-dependent differences in cold sensitivity appeared at the end of oil production. Genotype-dependent differences in FAD2.1, FAD2.2, FAD6, and FAD7 expression levels occurred in the epi-mesocarp cells during the oleogenic period. However, FAD2.1 and FAD7 were always the highest in the first part of this period. FAD2.2 and FAD7 increased after cold applications during oleogenesis, independently of the genotype. Unsaturated fatty acids increased in the drupes of the non-hardy genotype, but not in those of the hardy one, after cold exposure at the time of the highest FAD transcription. The results show a direct relationship between FAD expression and lipid desaturation in the drupes of the cold-sensitive genotype, and an inverse relationship in those of the cold-resistant genotype, suggesting that drupe cold acclimation requires a fine FAD post-transcriptional regulation. Hypotheses relating FAD desaturation to storage and membrane lipids, and genotype cold hardiness are discussed. PMID:21357772

Genome-Wide Survey and Characterization of Fatty Acid Desaturase Gene Family in Brassica napus and Its Parental Species.

PubMed

Xue, Yufei; Chen, Baojun; Wang, Rui; Win, Aung Naing; Li, Jiana; Chai, Yourong

2018-02-01

Rapeseed (Brassica napus) is an important oilseed crop worldwide, and fatty acid (FA) compositions determine the nutritional and economic value of its seed oil. Fatty acid desaturases (FADs) play a pivotal role in regulating FA compositions, but to date, no comprehensive genome-wide analysis of FAD gene family in rapeseed and its parent species has been reported. In this study, using homology searches, 84, 45, and 44 FAD genes were identified in rapeseed, Brassica rapa, and Brassica oleracea genomes, respectively. These FAD genes were unevenly located in 17 chromosomes and 2 scaffolds of rapeseed, 9 chromosomes and 1 scaffold of B. rapa, and all the chromosomes of B. oleracea. Phylogenetic analysis showed that the soluble and membrane-bound FADs in the three Brassica species were divided into four and six subfamilies, respectively. Generally, the soluble FADs contained two conserved histidine boxes, while three highly conserved histidine boxes were harbored in membrane-bound FADs. Exon-intron structure, intron phase, and motif composition and position were highly conserved in each FAD subfamily. Putative subcellular locations of FAD proteins in three Brassica species were consistent with those of corresponding known FADs. In total, 25 of simple sequence repeat (SSR) loci were found in FAD genes of the three Brassica species. Transcripts of selected FAD genes in the three species were examined in various organs/tissues or stress treatments from NCBI expressed sequence tag (EST) database. This study provides a critical molecular basis for quality improvement of rapeseed oil and facilitates our understanding of key roles of FAD genes in plant growth and development and stress response.

Thioredoxin System from Deinococcus radiodurans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Obiero, Josiah; Pittet, Vanessa; Bonderoff, Sara A.

2010-05-03

This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5{prime}-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-{angstrom} resolution. The overallmore » structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K{sub m} (5.7 {micro}M) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K{sub m}, 44.4 {micro}M). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.« less

Functional desaturase Fads1 (Δ5) and Fads2 (Δ6) orthologues evolved before the origin of jawed vertebrates.

PubMed

Castro, Luís Filipe Costa; Monroig, Óscar; Leaver, Michael J; Wilson, Jonathan; Cunha, Isabel; Tocher, Douglas R

2012-01-01

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

Variants in CPT1A, FADS1, and FADS2 are Associated with Higher Levels of Estimated Plasma and Erythrocyte Delta-5 Desaturases in Alaskan Eskimos.

PubMed

Voruganti, V Saroja; Higgins, Paul B; Ebbesson, Sven O E; Kennish, John; Göring, Harald H H; Haack, Karin; Laston, Sandra; Drigalenko, Eugene; Wenger, Charlotte R; Harris, William S; Fabsitz, Richard R; Devereux, Richard B; Maccluer, Jean W; Curran, Joanne E; Carless, Melanie A; Johnson, Matthew P; Moses, Eric K; Blangero, John; Umans, Jason G; Howard, Barbara V; Cole, Shelley A; Comuzzie, Anthony Gean

2012-01-01

The delta-5 and delta-6 desaturases (D5D and D6D), encoded by fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes, respectively, are rate-limiting enzymes in the metabolism of ω-3 and ω-6 fatty acids. The objective of this study was to identify genes influencing variation in estimated D5D and D6D activities in plasma and erythrocytes in Alaskan Eskimos (n = 761) participating in the genetics of coronary artery disease in Alaska Natives (GOCADAN) study. Desaturase activity was estimated by product: precursor ratio of polyunsaturated fatty acids. We found evidence of linkage for estimated erythrocyte D5D (eD5D) on chromosome 11q12-q13 (logarithm of odds score = 3.5). The confidence interval contains candidate genes FADS1, FADS2, 7-dehydrocholesterol reductase (DHCR7), and carnitine palmitoyl transferase 1A, liver (CPT1A). Measured genotype analysis found association between CPT1A, FADS1, and FADS2 single-nucleotide polymorphisms (SNPs) and estimated eD5D activity (p-values between 10(-28) and 10(-5)). A Bayesian quantitative trait nucleotide analysis showed that rs3019594 in CPT1A, rs174541 in FADS1, and rs174568 in FADS2 had posterior probabilities > 0.8, thereby demonstrating significant statistical support for a functional effect on eD5D activity. Highly significant associations of FADS1, FADS2, and CPT1A transcripts with their respective SNPs (p-values between 10(-75) and 10(-7)) in Mexican Americans of the San Antonio Family Heart Study corroborated our results. These findings strongly suggest a functional role for FADS1, FADS2, and CPT1A SNPs in the variation in eD5D activity.

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

PubMed

Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

2016-01-01

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

DNA methylation in an enhancer region of the FADS cluster is associated with FADS activity in human liver.

PubMed

Howard, Timothy D; Mathias, Rasika A; Seeds, Michael C; Herrington, David M; Hixson, James E; Shimmin, Lawrence C; Hawkins, Greg A; Sellers, Matthew; Ainsworth, Hannah C; Sergeant, Susan; Miller, Leslie R; Chilton, Floyd H

2014-01-01

Levels of omega-6 (n-6) and omega-3 (n-3), long chain polyunsaturated fatty acids (LcPUFAs) such as arachidonic acid (AA; 20:4, n-6), eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) impact a wide range of biological activities, including immune signaling, inflammation, and brain development and function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1) are rate limiting in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs) such as LA (18:2, n-6) to AA and α-linolenic acid (ALA, 18:3, n-3) to EPA and DHA. GWAS and candidate gene studies have consistently identified genetic variants within FADS1 and FADS2 as determinants of desaturase efficiencies and levels of LcPUFAs in circulating, cellular and breast milk lipids. Importantly, these same variants are documented determinants of important cardiovascular disease risk factors (total, LDL, and HDL cholesterol, triglycerides, CRP and proinflammatory eicosanoids). FADS1 and FADS2 lie head-to-head (5' to 5') in a cluster configuration on chromosome 11 (11q12.2). There is considerable linkage disequilibrium (LD) in this region, where multiple SNPs display association with LcPUFA levels. For instance, rs174537, located ∼ 15 kb downstream of FADS1, is associated with both FADS1 desaturase activity and with circulating AA levels (p-value for AA levels = 5.95 × 10(-46)) in humans. To determine if DNA methylation variation impacts FADS activities, we performed genome-wide allele-specific methylation (ASM) with rs174537 in 144 human liver samples. This approach identified highly significant ASM with CpG sites between FADS1 and FADS2 in a putative enhancer signature region, leading to the hypothesis that the phenotypic associations of rs174537 are likely due to methylation differences. In support of this hypothesis, methylation levels of the most significant probe were strongly associated with FADS1 and, to a lesser degree, FADS2 activities.

Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica

PubMed Central

2013-01-01

Background Crambe abyssinica produces high erucic acid (C22:1, 55-60%) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed. Results The individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5%) and PUFA reduction (by 8.6% to 10.2%). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P < 0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil. Conclusions CaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe. PMID:24083776

The Structure of the Flavoprotein Tryptophan-2-Monooxygenase, a Key Enzyme in the Formation of Galls in Plants†

PubMed Central

Gaweska, Helena M.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

2013-01-01

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic-acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from Pseudomonas savastanoi has been determined by X-ray diffraction methods to a resolution of 1.95 Å. The overall structure of the protein shows that it has the same fold as the monoamine oxidase family of flavoproteins, with the greatest similarities to the L-amino acid oxidases. The location of bound indole-3-acetamide in the active site enables identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the kcat and kcat/KTrp values by 60,000 and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, consistent with carbon-hydrogen bond cleavage becoming rate-limiting in the mutant enzyme. The W466F mutation decreases the kcat value less than 2-fold and the kcat/KTrp value only 5-fold, while the W466M mutation results in enzyme lacking flavin and detectable activity. This is consistent with a role for Trp466 in maintaining the structure of the flavin binding site in the more conserved FAD domain. PMID:23521653

Facebook Addiction Disorder (FAD) among German students—A longitudinal approach

PubMed Central

Margraf, Jürgen

2017-01-01

The present study aimed to investigate Facebook Addiction Disorder (FAD) in a German student sample over a period of one year. While mean FAD level did not increase during the investigation period, a significant increase was shown in the number of participants reaching the critical cutoff score. FAD was significantly positively related to the personality trait narcissism and to negative mental health variables (depression, anxiety, and stress symptoms). Furthermore, FAD fully mediated the significant positive relationship between narcissism and stress symptoms, which demonstrates that narcissistic people can be specifically at risk to develop FAD. Present results give a first overview of FAD in Germany. Practical applications for future studies and limitations of present results are discussed. PMID:29240823

Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.

PubMed

Ozawa, Kazumichi; Iwasa, Hisanori; Sasaki, Noriko; Kinoshita, Nao; Hiratsuka, Atsunori; Yokoyama, Kenji

2017-01-01

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m ) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.

Molecular variation of the nonribosomal peptide-polyketide siderophore yersiniabactin through biosynthetic and metabolic engineering.

PubMed

Ahmadi, Mahmoud Kamal; Fawaz, Samar; Fang, Lei; Yu, Zhipeng; Pfeifer, Blaine A

2016-05-01

The production of the mixed nonribosomal peptide-polyketide natural product yersiniabactin (Ybt) has been established using E. coli as a heterologous host. In this study, precursor-directed biosynthesis was used to generate five new analogs of Ybt, demonstrating the flexibility of the heterologous system and the biosynthetic process in allowing compound diversity. A combination of biosynthetic and cellular engineering was then used to influence the production metrics of the resulting analogs. First, the cellular levels and activity of FadL, a hydrocarbon transport protein, were tested for subsequent influence upon exogenous precursor uptake and Ybt analog production with a positive correlation observed between FadL over-production and analog formation. Next, a Ybt biosynthetic editing enzyme was removed from the heterologous system which decreased native compound production but increased analog formation. A final series of experiments enhanced endogenous anthranilate towards complete pathway formation of the associated analog which showed a selective ability to bind gold. © 2015 Wiley Periodicals, Inc.

Identification of eukaryotic UDP-galactopyranose mutase inhibitors using the ThermoFAD assay.

PubMed

Martín Del Campo, Julia S; Eckshtain-Levi, Meital; Sobrado, Pablo

2017-11-04

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of a pneumatic high-angle-of-attack Flush Airdata Sensing (HI-FADS) system

NASA Technical Reports Server (NTRS)

Whitmore, Stephen A.; Moes, Timothy R.; Leondes, Cornelius T.

1992-01-01

The HI-FADS system design is an evolution of the FADS systems (e.g., Larson et al., 1980, 1987), which emphasizes the entire airdata system development. This paper describes the HI-FADS measurement system, with particular consideration given to the basic measurement hardware and the development of the HI-FADS aerodynamic model and the basic nonlinear regression algorithm. Algorithm initialization techniques are developed, and potential algorithm divergence problems are discussed. Data derived from HI-FADS flight tests are used to demonstrate the system accuracies and to illustrate the developed concepts and methods.

Inverse Flush Air Data System (FADS) for Real Time Simulations

NASA Astrophysics Data System (ADS)

Madhavanpillai, Jayakumar; Dhoaya, Jayanta; Balakrishnan, Vidya Saraswathi; Narayanan, Remesh; Chacko, Finitha Kallely; Narayanan, Shyam Mohan

2017-12-01

Flush Air Data Sensing System (FADS) forms a mission critical sub system in future reentry vehicles. FADS makes use of surface pressure measurements from the nose cap of the vehicle for deriving the air data parameters of the vehicle such as angle of attack, angle of sideslip, Mach number, etc. These parameters find use in the flight control and guidance systems, and also assist in the overall mission management. The FADS under consideration in this paper makes use of nine pressure ports located in the nose cap of a technology demonstrator vehicle. In flight, the air data parameters are obtained from the FADS estimation algorithm using the pressure data at the nine pressure ports. But, these pressure data will not be available, for testing the FADS package during ground simulation. So, an inverse software to FADS which estimates the pressure data at the pressure ports for a given flight condition is developed. These pressure data at the nine ports will go as input to the FADS package during ground simulation. The software is run to generate the pressure data for the descent phase trajectory of the technology demonstrator. This data is used again to generate the air data parameters from FADS algorithm. The computed results from FADS algorithm match well with the trajectory data.

Hydrogen Sulfide Sensing through Reactive Sulfur Species (RSS) and Nitroxyl (HNO) in Enterococcus faecalis.

PubMed

Shen, Jiangchuan; Walsh, Brenna J C; Flores-Mireles, Ana Lidia; Peng, Hui; Zhang, Yifan; Zhang, Yixiang; Trinidad, Jonathan C; Hultgren, Scott J; Giedroc, David P

2018-05-17

Recent studies of hydrogen sulfide (H 2 S) signaling implicate low molecular weight (LMW) thiol persulfides and other reactive sulfur species (RSS) as signaling effectors. Here, we show that a CstR protein from the human pathogen Enterococcus faecalis ( E. faecalis), previously identified in Staphylococcus aureus ( S. aureus), is an RSS-sensing repressor that transcriptionally regulates a cst-like operon in response to both exogenous sulfide stress and Angeli's salt, a precursor of nitroxyl (HNO). E. faecalis CstR reacts with coenzyme A persulfide (CoASSH) to form interprotomer disulfide and trisulfide bridges between C32 and C61', which negatively regulate DNA binding to a consensus CstR DNA operator. A Δ cstR strain exhibits deficiency in catheter colonization in a catheter-associated urinary tract infection (CAUTI) mouse model, suggesting sulfide regulation and homeostasis is critical for pathogenicity. Cellular polysulfide metabolite profiling of sodium sulfide-stressed E. faecalis confirms an increase in both inorganic polysulfides and LMW thiols and persulfides sensed by CstR. The cst-like operon encodes two authentic thiosulfate sulfurtransferases and an enzyme we characterize here as an NADH and FAD-dependent coenzyme A (CoA) persulfide reductase (CoAPR) that harbors an N-terminal CoA disulfide reductase (CDR) domain and a C-terminal rhodanese homology domain (RHD). Both cysteines in the CDR (C42) and RHD (C508) domains are required for CoAPR activity and complementation of a sulfide-induced growth phenotype of a S. aureus strain lacking cstB, encoding a nonheme Fe II persulfide dioxygenase. We propose that S. aureus CstB and E. faecalis CoAPR employ orthogonal chemistries to lower CoASSH that accumulates under conditions of cellular sulfide toxicity and signaling.

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

PubMed

Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Rui-Fen; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

2013-04-01

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

PubMed

Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

2008-05-01

Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil.

PubMed

Pham, Anh-Tung; Shannon, J Grover; Bilyeu, Kristin D

2012-08-01

High oleic acid soybeans were produced by combining mutant FAD2-1A and FAD2-1B genes. Despite having a high oleic acid content, the linolenic acid content of these soybeans was in the range of 4-6 %, which may be high enough to cause oxidative instability of the oil. Therefore, a study was conducted to incorporate one or two mutant FAD3 genes into the high oleic acid background to further reduce the linolenic acid content. As a result, soybean lines with high oleic acid and low linolenic acid (HOLL) content were produced using different sources of mutant FAD2-1A genes. While oleic acid content of these HOLL lines was stable across two testing environments, the reduction of linolenic acid content varied depending on the number of mutant FAD3 genes combined with mutant FAD2-1 genes, on the severity of mutation in the FAD2-1A gene, and on the testing environment. Combination of two mutant FAD2-1 genes and one mutant FAD3 gene resulted in less than 2 % linolenic acid content in Portageville, Missouri (MO) while four mutant genes were needed to achieve the same linolenic acid in Columbia, MO. This study generated non-transgenic soybeans with the highest oleic acid content and lowest linolenic acid content reported to date, offering a unique alternative to produce a fatty acid profile similar to olive oil.

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Comparison of clinical characteristics between familial and non-familial early onset Alzheimer's disease.

PubMed

Joshi, Aditi; Ringman, John M; Lee, Albert S; Juarez, Kevin O; Mendez, Mario F

2012-10-01

Although familial Alzheimer's disease (FAD) is an early onset AD (EAD), most patients with EAD do not have a familial disorder. Recent guidelines recommend testing for genes causing FAD only in those EAD patients with two first-degree relatives. However, some patients with FAD may lack a known family history or other indications for suspecting FAD but might nonetheless be carriers of FAD mutations. The study was aimed to identify clinical features that distinguish FAD from non-familial EAD (NF-EAD). A retrospective review of a university-based cohort of 32 FAD patients with PSEN1-related AD and 81 with NF-EAD was conducted. The PSEN1 patients, compared to the NF-EAD patients, had an earlier age of disease onset (41.8 ± 5.2 vs. 55.9 ± 4.8 years) and, at initial assessment, a longer disease duration (5.1 ± 3.4 vs. 3.3 ± 2.6 years) and lower MMSE scores (10.74 ± 8.0 vs. 20.95 ± 5.8). Patients with NF-EAD were more likely to present with non-memory deficits, particularly visuospatial symptoms, than were FAD patients. When age, disease duration, and MMSE scores were controlled in a logistical regression model, FAD patients were more likely to have significant headaches, myoclonus, gait abnormality, and pseudobulbar affect than those with NF-EAD. In addition to a much younger age of onset, FAD patients with PSEN1 mutations differed from those with NF-EAD by a history of headaches and pseudobulbar affect, as well as myoclonus and gait abnormality on examination. These may represent differences in pathophysiology between FAD and NF-EAD and in some contexts such findings should lead to genetic counseling and appropriate recommendations for genetic testing for FAD.

Comparison of clinical characteristics between familial and non-familial early onset Alzheimer’s disease

PubMed Central

Ringman, John M.; Lee, Albert S.; Juarez, Kevin O.; Mendez, Mario F.

2012-01-01

Although familial Alzheimer’s disease (FAD) is an early onset AD (EAD), most patients with EAD do not have a familial disorder. Recent guidelines recommend testing for genes causing FAD only in those EAD patients with two first-degree relatives. However, some patients with FAD may lack a known family history or other indications for suspecting FAD but might nonetheless be carriers of FAD mutations. The study was aimed to identify clinical features that distinguish FAD from non-familial EAD (NF-EAD). A retrospective review of a university-based cohort of 32 FAD patients with PSEN1-related AD and 81 with NF-EAD was conducted. The PSEN1 patients, compared to the NF-EAD patients, had an earlier age of disease onset (41.8 ± 5.2 vs. 55.9 ± 4.8 years) and, at initial assessment, a longer disease duration (5.1 ± 3.4 vs. 3.3 ± 2.6 years) and lower MMSE scores (10.74 ± 8.0 vs. 20.95 ± 5.8). Patients with NF-EAD were more likely to present with non-memory deficits, particularly visuospatial symptoms, than were FAD patients. When age, disease duration, and MMSE scores were controlled in a logistical regression model, FAD patients were more likely to have significant headaches, myoclonus, gait abnormality, and pseudobulbar affect than those with NF-EAD. In addition to a much younger age of onset, FAD patients with PSEN1 mutations differed from those with NF-EAD by a history of headaches and pseudobulbar affect, as well as myoclonus and gait abnormality on examination. These may represent differences in pathophysiology between FAD and NF-EAD and in some contexts such findings should lead to genetic counseling and appropriate recommendations for genetic testing for FAD. PMID:22460587

Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.

PubMed

Martínez, Eriel; Estupiñán, Mónica; Pastor, F I Javier; Busquets, Montserrat; Díaz, Pilar; Manresa, Angeles

2013-02-01

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Dietary arachidonic acid and docosahexaenoic acid regulate liver fatty acid desaturase (FADS) alternative transcript expression in suckling piglets.

PubMed

Wijendran, Vasuki; Downs, Ian; Srigley, Cynthia Tyburczy; Kothapalli, Kumar S D; Park, Woo Jung; Blank, Bryant S; Zimmer, J Paul; Butt, C M; Salem, Norman; Brenna, J Thomas

2013-10-01

Molecular regulation of fatty acid desaturase (Fads) gene expression by dietary arachidonic acid (ARA) and docosahexaenoic acid (DHA) during early post-natal period, when the demand for long chain polyunsaturated fatty acids (LC-PUFA) is very high, has not been well defined. The objective of the current study was to determine regulation of liver Fads1, Fads2 and Fads3 classical (CS) and alternative transcripts (AT) expression by dietary ARA and DHA, within the physiological range present in human breast milk, in suckling piglets. Piglets were fed one of six milk replacer formula diets (formula-reared groups, FR) with varying ARA and DHA content from days 3-28 of age. The ARA/DHA levels of the six formula diets were as follows (% total fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. The control maternal-reared (MR) group remained with the dam. Fads1 expression was not significantly different between FR and MR groups. Fads2 expression was down-regulated significantly in diets with 1:1 ratio of ARA:DHA, compared to MR. Fads2 AT1 expression was highly correlated to Fads2 expression. Fads3 AT7 was the only Fads3 transcript sensitive to dietary LC-PUFA intake and was up-regulated in the formula diets with lowest ARA and DHA contents compared to MR. Thus, the present study provides evidence that the proportion of dietary ARA:DHA is a significant determinant of Fads2 expression and LC-PUFA metabolism during the early postnatal period. Further, the data suggest that Fads3 AT7 may have functional significance when dietary supply of ARA and DHA are low during early development. © 2013 Elsevier Ltd. All rights reserved.

A novel FAD2-1 A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content.

PubMed

Pham, Anh-Tung; Lee, Jeong-Dong; Shannon, J Grover; Bilyeu, Kristin D

2011-09-01

The alteration of fatty acid profiles in soybean to improve soybean oil quality has been a long-time goal of soybean researchers. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In the lipid biosynthetic pathway, the enzyme fatty acid desaturase 2 (FAD2) is responsible for the conversion of oleic acid precursors to linoleic acid precursors in developing soybean seeds. Two genes encoding FAD2-1A and FAD2-1B were identified to be expressed specifically in seeds during embryogenesis and have been considered to hold an important role in controlling the seed oleic acid content. A total of 22 soybean plant introduction (PI) lines identified to have an elevated oleic acid content were characterized for sequence mutations in the FAD 2-1A and FAD2-1B genes. PI 603452 was found to contain a deletion of a nucleotide in the second exon of FAD2-1A. These important SNPs were used in developing molecular marker genotyping assays. The assays appear to be a reliable and accurate tool to identify the FAD 2-1A and FAD2-1B genotype of wild-type and mutant plants. PI 603452 was subsequently crossed with PI 283327, a soybean line that has a mutation in FAD2-1B. Interestingly, soybean lines carrying both homozygous insertion/deletion mutation (indel) FAD2-1A alleles and mutant FAD2-1B alleles have an average of 82-86% oleic acid content, compared to 20% in conventional soybean, and low levels of linoleic and linolenic acids. The newly identified indel mutation in the FAD2-1A gene offers a simple method for the development of high oleic acid commercial soybean varieties.

Blocking phosphatidylcholine utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the importance of this nutrient source in vivo.

PubMed

Sun, Zhenxin; Kang, Yun; Norris, Michael H; Troyer, Ryan M; Son, Mike S; Schweizer, Herbert P; Dow, Steven W; Hoang, Tung T

2014-01-01

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

Combined deficiency of iron and (n-3) fatty acids in male rats disrupts brain monoamine metabolism and produces greater memory deficits than iron deficiency or (n-3) fatty acid deficiency alone.

PubMed

Baumgartner, Jeannine; Smuts, Cornelius M; Malan, Linda; Arnold, Myrtha; Yee, Benjamin K; Bianco, Laura E; Boekschoten, Mark V; Müller, Michael; Langhans, Wolfgang; Hurrell, Richard F; Zimmermann, Michael B

2012-08-01

Deficiencies of iron (Fe) (ID) and (n-3) fatty acids (FA) [(n-3)FAD] may impair brain development and function through shared mechanisms. However, little is known about the potential interactions between these 2 common deficiencies. We studied the effects of ID and (n-3)FAD, alone and in combination, on brain monoamine pathways (by measuring monoamines and related gene expression) and spatial working and reference memory (by Morris water maze testing). Using a 2 × 2 design, male rats were fed an ID, (n-3)FAD, ID+(n-3)FAD, or control diet for 5 wk postweaning (postnatal d 21-56) after (n-3)FAD had been induced over 2 generations. The (n-3)FAD and ID diets decreased brain (n-3) FA by 70-76% and Fe by 20-32%, respectively. ID and (n-3)FAD significantly increased dopamine (DA) concentrations in the olfactory bulb (OB) and striatum, with an additive 1- to 2-fold increase in ID+(n-3)FAD rats compared with controls (P < 0.05). ID decreased serotonin (5-HT) levels in OB, with a significant decrease in ID+(n-3)FAD rats. Furthermore, norepinephrine concentrations were increased 2-fold in the frontal cortex (FC) of (n-3)FAD rats (P < 0.05). Dopa decarboxylase was downregulated in the hippocampus of ID and ID+(n-3)FAD rats (fold-change = -1.33; P < 0.05). ID and (n-3)FAD significantly impaired working memory performance and the impairment positively correlated with DA concentrations in FC (r = 0.39; P = 0.026). Reference memory was impaired in the ID+(n-3)FAD rats (P < 0.05) and was negatively associated with 5-HT in FC (r = -0.42; P = 0.018). These results suggest that the combined deficiencies of Fe and (n-3) FA disrupt brain monoamine metabolism and produce greater deficits in reference memory than ID or (n-3)FAD alone.

Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH).

PubMed

Gupta, Ashish; Popowich, Shelly; Ojkic, Davor; Kurukulasuriya, Shanika; Chow-Lockerbie, Betty; Gunawardana, Thushari; Goonewardene, Kalhari; Karunarathna, Ruwani; Ayalew, Lisanework E; Ahmed, Khawaja Ashfaque; Tikoo, Suresh K; Willson, Philip; Gomis, Susantha

2018-01-29

Fowl adenovirus (FAdV) is comprised of five species (A to E) and 12 serotypes (1-7, 8a, 8b, 9-11). Inclusion body hepatitis (IBH) is caused by FAdV-7, 8a, 8b (species E) and FAdV-2 and 11 (species D). Commercial vaccines against IBH are not available in Canada. Autogenous FAdV broiler breeder vaccines are now used in some areas where outbreaks of IBH are occurring. The objective of this study was to evaluate the efficacy of a bivalent (species D and E) live and an inactivated FAdV broiler breeder vaccine in protecting broiler chicks against IBH through maternal antibody (MtAb) transfer. FAdV seronegative broiler breeders (n = 300/group) received either a live or inactivated bivalent (FAdV-8b-SK + FAdV-11-1047) vaccine. The live vaccine (1 × 10 4 TCID 50 of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 10 6 TCID 50 of each virus + 20% Emulsigen D) was given intramuscularly at 16 and 19 weeks of age. Controls (n = 150) were given saline orally. The inactivated vaccine group was boosted 3 weeks later with the same vaccine. Neutralizing antibodies (NAb) in sera (n = 10) were detected at 19, 22, 30 and 48 weeks of age. NAb were able to neutralize various FAdV serotypes within species D and E. Mean NAb were similar in the both live and killed vaccine groups at 19, 30 and 48 weeks and ranged from 2.4 to 3.7 log 10 . Approximately 26 ± 7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 10 7 TCID 50 /bird intramuscularly) of FAdV-8b-SK, FAdV-11-1047, or FAdV-2-685 (n = 90/group) at 14 days post-hatch (dph) showed 98-100% protection in broiler chicks to homologous or heterologous FAdV challenges. Our data suggests that a bivalent live and an inactivated FAdV vaccine are equally effective and have the potential for the control of IBH. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protection against RAGE-mediated neuronal cell death by sRAGE-secreting human mesenchymal stem cells in 5xFAD transgenic mouse model.

PubMed

Son, Myeongjoo; Oh, Seyeon; Park, Hyunjin; Ahn, Hyosang; Choi, Junwon; Kim, Hyungho; Lee, Hye Sun; Lee, Sojung; Park, Hye-Jeong; Kim, Seung U; Lee, Bonghee; Byun, Kyunghee

2017-11-01

Alzheimer's disease (AD), which is the most commonly encountered neurodegenerative disease, causes synaptic dysfunction and neuronal loss due to various pathological processes that include tau abnormality and amyloid beta (Aβ) accumulation. Aβ stimulates the secretion and the synthesis of Receptor for Advanced Glycation End products (RAGE) ligand by activating microglial cells, and has been reported to cause neuronal cell death in Aβ 1-42 treated rats and in mice with neurotoxin-induced Parkinson's disease. The soluble form of RAGE (sRAGE) is known to reduce inflammation, and to decrease microglial cell activation and Aβ deposition, and thus, it protects from neuronal cell death in AD. However, sRAGE protein has too a short half-life for therapeutic purposes. We developed sRAGE-secreting umbilical cord derived mesenchymal stem cells (sRAGE-MSCs) to enhance the inhibitory effects of sRAGE on Aβ deposition and to reduce the secretion and synthesis of RAGE ligands in 5xFAD mice. In addition, these cells improved the viability of injected MSCs, and enhanced the protective effects of sRAGE by inhibiting the binding of RAGE and RAGE ligands in 5xFAD mice. These findings suggest sRAGE protein from sRAGE-MSCs has better protection against neuronal cell death than sRAGE protein or single MSC treatment by inhibiting the RAGE cell death cascade and RAGE-induce inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

Setting the Record Straight. The Truth About Fad Diets.

ERIC Educational Resources Information Center

Wheat Foods Council, Parker, CO.

The Setting the Record Straight information packet presents facts to set the record straight about nutrition and debunk fad diets. The kit features materials designed to communicate the importance of balanced eating. Materials include: a time line of fad diets; four reproducible fad diet book review handouts that show the misleading claims rampant…

Fowl Adenoviruses D and E Cause Inclusion Body Hepatitis Outbreaks in Broiler and Broiler Breeder Pullet Flocks.

PubMed

Morshed, Rima; Hosseini, Hossein; Langeroudi, Arash Ghalyanchi; Fard, Mohammad Hassan Bozorgmehri; Charkhkar, Saeid

2017-06-01

Twenty-four fowl adenoviruses (FAdVs) were isolated from broiler and broiler breeder pullet flocks in Iran during 2013-2016 and were identified and characterized. All FAdVs were from inclusion body hepatitis (IBH) cases, showing an enlarged and pale yellow liver with multiple petechial hemorrhages. Phylogenetic analyses of partial hexon gene sequences are an adequate and quick method for differentiation and genotyping. The isolates were subjected to PCR to amplify a 590-bp fragment from the hexon gene. Sequence analysis revealed the presence of two species D and E. Eighty FAdV isolates were genetically related to the strain EU979378 of FAdV-11 (96.5% to 97.6% identity), and six isolates were related to the strain EU979375 of FAdV-8b (97% identity). The results indicated that two FAdV serotypes (11 and 8b) are high prevalence serotypes of FAdVs in Iran and are pathogenic enough to cause IBH in young chicks. Therefore, preventive measures against FAdV infection on poultry farms should be implemented.

[Mutations of amyloid precursor protein in early-onset familial Alzheimer's disease].

PubMed

Naruse, S; Tsuji, S; Miyatake, T

1992-09-01

Genetic linkage studies of familial Alzheimer's disease (FAD) have suggested that some form of early-onset FAD is linked to proximal long arm of chromosome 21. It has been also suggested that some form of late-onset FAD is linked to long arm of chromosome 19. Goate et al have identified a mis-sense mutation (Val to Ile) in exon 17 of the amyloid precursor protein (APP) gene in 2 of 16 early-onset FAD families, and have shown that the FAD locus in an FAD family is tightly linked to the mis-sense mutation. To determine if the mis-sense mutation is observed in different ethnic origine, we have studied some early-onset FAD families. Two early-onset FAD families showed the existence of the mutation. As the mutation has been identified in different ethnic origine and the mutation has not been observed in normal individuals, it strengthen hypothesis that the mutation is pathogenic. Recently, Val to Phe and Val to Gly mutations have been also identified at the same codon (Codon 717) of the APP gene.

Biosynthesis of Polyunsaturated Fatty Acids in the Razor Clam Sinonovacula constricta: Characterization of Δ5 and Δ6 Fatty Acid Desaturases.

PubMed

Ran, Zhaoshou; Xu, Jilin; Liao, Kai; Li, Shuang; Chen, Shubing; Yan, Xiaojun

2018-05-09

To investigate the endogenous long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic ability in Sinonovacula constricta, fatty acid desaturases (Fads) of this bivalve, namely, Scfad5a, Scfad5b, and Scfad6, were cloned and characterized in the current study. Meanwhile, the tissue distributions of S. constricta Fads and fatty acids (FAs) were examined. Heterologous expression in yeasts confirmed that Scfad5a and Scfad5b were both Δ5 Fads, while Scfad6 was a Δ6 Fad. However, compared with Fads in other organisms, the desaturation activities of S. constricta Fads were relatively low (especially for Scfad6), indicating an adaptation to living conditions. S. constricta Fads were expressed in all tissues examined, and particularly high expressions were found in intestine and gonad. Moreover, FAs were differently distributed among tissues, which might be correlated with their corresponding physiological roles. Taken together, the results provided an insight into LC-PUFA biosynthesis in S. constricta. Notably, Scfad6 was the first functionally characterized Δ6 Fad in marine molluscs to date.

Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China.

PubMed

Zhang, Teng; Jin, Qianyue; Ding, Peiyang; Wang, Yinbiao; Chai, Yongxiao; Li, Yafei; Liu, Xiao; Luo, Jun; Zhang, Gaiping

2016-11-18

In several parts of China, there have been a large number of hydropericardium syndrome (HPS) outbreaks caused by serotype 4 fowl adenovirus (FAdV-4) in broiler chickens since 2015. These outbreak-associated FAdV-4 strains were distinct from previous circulating strains which did not lead to severe HPS outbreaks. To better understand the molecular epidemiology of the currently circulating FAdV strains for effective diagnosis and treatment of HPS, we isolated 12 HPS outbreak-associated FAdV-4 strains from different regions in central China and investigated their molecular characteristics by performing phylogenetic analyses based on the hexon genes. Our results indicated the FAdV-4 strains in this study all belonged to serotype FAdV-4, species FAdV-C. And in comparison with ON1, KR5, MX-SHP95, PK-01, PJ-06 strains within the cluster where outbreak-associated FAdV-4 strains were located, the nucleotide sequence divergence were 1.31, 1.10, 1.42, 2.77 and 2.84%, respectively. Phylogenetic analyses revealed the hexon genes of the 12 outbreak-associated strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor and we suggested that these outbreak-associated FAdV-4 strains originate from earlier strains in India.

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

PubMed

Vasilchenko, Liliya G; Ludwig, Roland; Yershevich, Olga P; Haltrich, Dietmar; Rabinovich, Mikhail L

2012-07-01

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Association of polyunsaturated fatty acids in breast milk with fatty acid desaturase gene polymorphisms among Chinese lactating mothers.

PubMed

Ding, Zhen; Liu, Guo-Liang; Li, Xiang; Chen, Xue-Yan; Wu, Yi-Xia; Cui, Can-Can; Zhang, Xi; Yang, Guang; Xie, Lin

2016-06-01

The fatty acid desaturase (FADS) controls polyunsaturated fatty acid (PUFA) synthesis in human tissues and breast milk. Evaluate the influence of 10 single nucleotide polymorphisms (SNPs) and various haplotypes in the FADS gene cluster (FADS1, FADS2, FADS3) on PUFA concentration in the breast milk of 209 healthy Chinese women. PUFA concentrations were measured in breast milk using gas chromatography and genotyping was performed using the Sequenom Mass Array system. A SNP (rs1535) and 2-locus haplotypes (rs3834458-rs1535, rs1535-rs174575) in the FADS2 gene were associated with concentrations of γ-linoleic acid (GLA) and arachidonic acid (AA) in breast milk. Likewise, in the FADS1 gene, a 2-locus constructed haplotype (rs174547-rs174553) also affected GLA and AA concentration (P<0.05 for all). Minor allele carriers of the SNP and haplotypes described above had lower concentrations of GLA and AA. In the FADS2 gene, the 3-locus haplotype rs3834458-rs1535-rs174575, significantly affected concentrations of GLA but not AA. Pairwise comparison showed that individuals major homozygous for the SNP rs1000778 in the FADS3 gene had lower concentrations of ALA and linoleic acid (LA) in their breast milk. Polymorphisms in the FADS gene cluster influence PUFA concentrations in the breast milk of Chinese Han lactating women. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aldosterone synthase inhibition improves cardiovascular function and structure in rats with heart failure: a comparison with spironolactone.

PubMed

Mulder, Paul; Mellin, Virginie; Favre, Julie; Vercauteren, Magali; Remy-Jouet, Isabelle; Monteil, Christelle; Richard, Vincent; Renet, Sylvanie; Henry, Jean Paul; Jeng, Arco Y; Webb, Randy L; Thuillez, Christian

2008-09-01

Inhibition of aldosterone synthase, the key enzyme in aldosterone formation, could be an alternative strategy for mineralocorticoid-receptor antagonists in congestive heart failure (CHF), but its effect in CHF is unknown. We compared, in rats with CHF, the effects of a 7 day and a 12 week treatment with the aldosterone synthase inhibitor FAD286 (4 mg kg(-1) day(-1)) with those induced by spironolactone (80 mg kg(-1) day(-1)). FAD286/spironolactone increased cardiac output without modifying arterial pressure. Long-term FAD286 and spironolactone reduced left ventricular (LV) end-diastolic pressure, LV relaxation constant, and LV dilatation, and these effects were more marked with FAD286, whereas both drugs reduced LV hypertrophy and collagen accumulation to the same extent. Long-term FAD286/spironolactone prevented CHF-related enhancement in LV ACE and reduction in LV ACE-2, but only FAD286 prevented the reduction in LV AT(2) receptors. FAD286, but not long-term spironolactone, reduced the CHF-related enhancements in LV reactive oxygen species, reduced-oxidized glutathione ratio, and aortic nicotinamide adenine dinucleotide phosphate oxidase activity. FAD286 normalized the CHF-induced impairment of endothelium-dependent vasodilatation. In experimental CHF, FAD286 and spironolactone improve LV haemodynamics, remodelling, and function, but only FAD286 persistently normalizes LV 'redox status'. These results suggest that aldosterone synthase inhibition is a potential therapeutic strategy for the treatment of CHF.

Germline mutations in RYR1 are associated with foetal akinesia deformation sequence/lethal multiple pterygium syndrome.

PubMed

McKie, Arthur B; Alsaedi, Atif; Vogt, Julie; Stuurman, Kyra E; Weiss, Marjan M; Shakeel, Hassan; Tee, Louise; Morgan, Neil V; Nikkels, Peter G J; van Haaften, Gijs; Park, Soo-Mi; van der Smagt, Jasper J; Bugiani, Marianna; Maher, Eamonn R

2014-12-05

Foetal akinesia deformation sequence syndrome (FADS) is a genetically heterogeneous disorder characterised by the combination of foetal akinesia and developmental defects which may include pterygia (joint webbing). Traditionally multiple pterygium syndrome (MPS) has been divided into two forms: prenatally lethal (LMPS) and non-lethal Escobar type (EVMPS) types. Interestingly, FADS, LMPS and EVMPS may be allelic e.g. each of these phenotypes may result from mutations in the foetal acetylcholine receptor gamma subunit gene (CHRNG). Many cases of FADS and MPS do not have a mutation in a known FADS/MPS gene and we undertook molecular genetic studies to identify novel causes of these phenotypes. After mapping a novel locus for FADS/LMPS to chromosome 19, we identified a homozygous null mutation in the RYR1 gene in a consanguineous kindred with recurrent LMPS pregnancies. Resequencing of RYR1 in a cohort of 66 unrelated probands with FADS/LMPS/EVMPS (36 with FADS/LMPS and 30 with EVMPS) revealed two additional homozygous mutations (in frame deletions). The overall frequency of RYR1 mutations in probands with FADS/LMPS was 8.3%. Our findings report, for the first time, a homozygous RYR1 null mutation and expand the range of RYR1-related phenotypes to include early lethal FADS/LMPS. We suggest that RYR1 mutation analysis should be performed in cases of severe FADS/LMPS even in the absence of specific histopathological indicators of RYR1-related disease.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

PubMed

Hofmann, Anja; Brunssen, Coy; Peitzsch, Mirko; Balyura, Mariya; Mittag, Jennifer; Frenzel, Annika; Jannasch, Anett; Brown, Nicholas F; Weldon, Steven M; Gueneva-Boucheva, Kristina K; Eisenhofer, Graeme; Bornstein, Stefan R; Morawietz, Henning

2017-06-01

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db / db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db / db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg / d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db / db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db / db mice. © Georg Thieme Verlag KG Stuttgart · New York.

Association of maternal weight with FADS and ELOVL genetic variants and fatty acid levels- The PREOBE follow-up

PubMed Central

de la Garza Puentes, Andrea; Montes Goyanes, Rosa; Chisaguano Tonato, Aida Maribel; Torres-Espínola, Francisco José; Arias García, Miriam; de Almeida, Leonor; Bonilla Aguirre, María; Guerendiain, Marcela; Castellote Bargalló, Ana Isabel; Segura Moreno, Maite; García-Valdés, Luz; Campoy, Cristina; Lopez-Sabater, M. Carmen

2017-01-01

Single nucleotide polymorphisms (SNPs) in the genes encoding the fatty acid desaturase (FADS) and elongase (ELOVL) enzymes affect long-chain polyunsaturated fatty acid (LC-PUFA) production. We aimed to determine if these SNPs are associated with body mass index (BMI) or affect fatty acids (FAs) in pregnant women. Participants (n = 180) from the PREOBE cohort were grouped according to pre-pregnancy BMI: normal-weight (BMI = 18.5–24.9, n = 88) and overweight/obese (BMI≥25, n = 92). Plasma samples were analyzed at 24 weeks of gestation to measure FA levels in the phospholipid fraction. Selected SNPs were genotyped (7 in FADS1, 5 in FADS2, 3 in ELOVL2 and 2 in ELOVL5). Minor allele carriers of rs174545, rs174546, rs174548 and rs174553 (FADS1), and rs1535 and rs174583 (FADS2) were nominally associated with an increased risk of having a BMI≥25. Only for the normal-weight group, minor allele carriers of rs174537, rs174545, rs174546, and rs174553 (FADS1) were negatively associated with AA:DGLA index. Normal-weight women who were minor allele carriers of FADS SNPs had lower levels of AA, AA:DGLA and AA:LA indexes, and higher levels of DGLA, compared to major homozygotes. Among minor allele carriers of FADS2 and ELOVL2 SNPs, overweight/obese women showed higher DHA:EPA index than the normal-weight group; however, they did not present higher DHA concentrations than the normal-weight women. In conclusion, minor allele carriers of FADS SNPs have an increased risk of obesity. Maternal weight changes the effect of genotype on FA levels. Only in the normal-weight group, minor allele carriers of FADS SNPs displayed reduced enzymatic activity and FA levels. This suggests that women with a BMI≥25 are less affected by FADS genetic variants in this regard. In the presence of FADS2 and ELOVL2 SNPs, overweight/obese women showed higher n-3 LC-PUFA production indexes than women with normal weight, but this was not enough to obtain a higher n-3 LC-PUFA concentration. PMID:28598979

Genetic variation in FADS genes is associated with maternal long-chain PUFA status but not with cognitive development of infants in a high fish-eating observational study.

PubMed

Yeates, Alison J; Love, Tanzy M; Engström, Karin; Mulhern, Maria S; McSorley, Emeir M; Grzesik, Katherine; Alhamdow, Ayman; Wahlberg, Karin; Thurston, Sally W; Davidson, Philip W; van Wijngaarden, Edwin; Watson, Gene E; Shamlaye, Conrad F; Myers, G J; Strain, J J; Broberg, Karin

2015-12-01

Long-chain n-6 and n-3 PUFA (LC-PUFA), arachidonic acid (AA) (20:4n-6) and DHA (22:6n-3), are critical for optimal brain development. These fatty acids can be consumed directly from the diet, or synthesized endogenously from precursor PUFA by Δ-5 (encoded by FADS1) and Δ-6 desaturases (encoded by FADS2). The aim of this study was to determine the potential importance of maternal genetic variability in FADS1 and FADS2 genes to maternal LC-PUFA status and infant neurodevelopment in populations with high fish intakes. The Nutrition Cohorts 1 (NC1) and 2 (NC2) are longitudinal observational mother-child cohorts in the Republic of Seychelles. Maternal serum LC-PUFA was measured at 28 weeks gestation and genotyping for rs174537 (FADS1), rs174561 (FADS1), rs3834458 (FADS1-FADS2) and rs174575 (FADS2) was performed in both cohorts. The children completed the Bayley Scales of Infant Development II (BSID-II) at 30 months in NC1 and at 20 months in NC2. Complete data were available for 221 and 1310 mothers from NC1 and NC2 respectively. With increasing number of rs3834458 minor alleles, maternal concentrations of AA were significantly decreased (NC1 p=0.004; NC2 p<0.001) and precursor:product ratios for linoleic acid (LA) (18:2n-6)-to-AA (NC1 p<0.001; NC2 p<0.001) and α-linolenic acid (ALA) (18:3n-3)-to-DHA were increased (NC2 p=0.028). There were no significant associations between maternal FADS genotype and BSID-II scores in either cohort. A trend for improved PDI was found among infants born to mothers with the minor rs3834458 allele.In these high fish-eating cohorts, genetic variability in FADS genes was associated with maternal AA status measured in serum and a subtle association of the FADS genotype was found with neurodevelopment. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.

The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species ( G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes ( FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes ( FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. These data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.« less

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

DOE PAGES

Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.; ...

2014-11-18

The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species ( G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes ( FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes ( FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. These data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.« less

Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding

PubMed Central

Ozdilek, Bagdeser A.; Thompson, Valery F.; Ahmed, Nasiha S.; White, Connor I.

2017-01-01

Abstract RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood. Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU. For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding. RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS. Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA. PMID:28575444

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members,more » implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.« less

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains.

PubMed

Teyra, Joan; Sidhu, Sachdev S; Kim, Philip M

2012-08-14

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains. Copyright © 2012. Published by Elsevier B.V.

Docosahexaenoic acid (DHA) supplementation in pregnancy differentially modulates arachidonic acid and DHA status across FADS genotypes in pregnancy.

PubMed

Scholtz, S A; Kerling, E H; Shaddy, D J; Li, S; Thodosoff, J M; Colombo, J; Carlson, S E

2015-03-01

Some FADS alleles are associated with lower DHA and ARA status assessed by the relative amount of arachidonic acid (ARA) and docosahexaenoic acid (DHA) in plasma and red blood cell (RBC) phospholipids (PL). We determined two FADS single nucleotide polymorphisms (SNPs) in a cohort of pregnant women and examined the relationship of FADS1rs174533 and FADS2rs174575 to DHA and ARA status before and after supplementation with 600mg per day of DHA. The 205 pregnant women studied were randomly assigned to placebo (mixed soy and corn oil) (n=96) or 600mg algal DHA (n=109) in 3 capsules per day for the last two trimesters of pregnancy. Women homozygous for the minor allele of FADS1rs174533 (but not FADS2rs174575) had lower DHA and ARA status at baseline. At delivery, minor allele homozygotes of FADS1rs174533 in the placebo group had lower RBC-DHA compared to major-allele carriers (P=0.031), while in the DHA-supplemented group, all genotypes had higher DHA status compared to baseline (P=0.001) and status did not differ by genotype (P=0.941). Surprisingly, DHA but not the placebo decreased ARA status of minor allele homozygotes of both FADS SNPs but not major allele homozygotes at delivery. Any physiological effects of changing the DHA to ARA ratio by increasing DHA intake appears to be greater in minor allele homozygotes of some FADS SNPs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

NASA Technical Reports Server (NTRS)

Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

1997-01-01

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

Application of the FADS system on the Re-entry Module

NASA Astrophysics Data System (ADS)

Zhen, Huang

2016-07-01

The aerodynamic model for Flush Air Data Sensing System (FADS) is built based on the surface pressure distribution obtained through the pressure orifices laid on specific positions of the surface,and the flight parameters,such as angle of attack,angle of side-slip,Mach number,free-stream static pressure and dynamic pressure are inferred from the aerodynamic model.The flush air data sensing system (FADS) has been used on several flight tests of aircraft and re-entry vehicle,such as,X-15,space shuttle,F-14,X-33,X-43A and so on. This paper discusses the application of the FADS on the re-entry module with blunt body to obtain high-precision aerodynamic parameters.First of all,a basic theory and operating principle of the FADS is shown.Then,the applications of the FADS on typical aircrafts and re-entry vehicles are described.Thirdly,the application mode on the re-entry module with blunt body is discussed in detail,including aerodynamic simulation,pressure distribution,trajectory reconstruction and the hardware shoule be used,such as flush air data sensing system(FADS),inertial navigation system (INS),data acquisition system,data storage system.Finally,ablunt module re-entry flight test from low earth orbit (LEO) is planned to obtain aerodynamic parameters and amend the aerodynamic model with this FADS system data.The results show that FADS system can be applied widely in re-entry module with blunt bodies.

Effect of a fuel activation device (FAD) on particulate matter and black carbon emissions from a diesel locomotive engine.

PubMed

Park, Duckshin; Lee, Taejeong; Lee, Yongil; Jeong, Wonseog; Kwon, Soon-Bark; Kim, Dongsool; Lee, Kiyoung

2017-01-01

Emission reduction is one of the most efficient control measures in fuel-powered locomotives. The purpose of this study was to determine the reduction in particulate matter (PM) and black carbon (BC) emissions following the installation of a fuel activation device (FAD). The FAD was developed to enhance fuel combustion by atomizing fuel and to increase the surface area per unit volume of injected fuel. Emission reduction by the FAD was evaluated by installing a FAD in an operating diesel locomotive in Mongolia. The test was conducted on a train operating on a round-trip 238-km route between Ulaanbaatar and Choir stations in Mongolia. The fuel consumption rate was slightly reduced following the FAD installation. The FAD installation decreased PM and BC emissions in the diesel locomotive, especially coarse PM. The PM 10 reductions achieved after FAD installation were 58.0, 69.7, and 34.2% for the constant velocity, stopping, and acceleration stages of the train's operation, respectively. The BC reduction rates were 29.5, 52.8, and 27.4% for the constant velocity, stopping, and acceleration stages, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

The prokaryotic FAD synthetase family: a potential drug target.

PubMed

Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

2013-01-01

Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

Conditional Deletion of Prnp Rescues Behavioral and Synaptic Deficits after Disease Onset in Transgenic Alzheimer's Disease

PubMed Central

Kaufman, Adam C.; Herber, Charlotte S.; Haas, Laura T.; Robinson, Sophie; Lee, Michael K.

2017-01-01

Biochemical and genetic evidence implicate soluble oligomeric amyloid-β (Aβo) in triggering Alzheimer's disease (AD) pathophysiology. Moreover, constitutive deletion of the Aβo-binding cellular prion protein (PrPC) prevents development of memory deficits in APPswe/PS1ΔE9 mice, a model of familial AD. Here, we define the role of PrPC to rescue or halt established AD endophenotypes in a therapeutic disease-modifying time window after symptom onset. Deletion of Prnp at either 12 or 16 months of age fully reverses hippocampal synapse loss and completely rescues preexisting behavioral deficits by 17 months. In contrast, but consistent with a neuronal function for Aβo/PrPC signaling, plaque density, microgliosis, and astrocytosis are not altered. Degeneration of catecholaminergic neurons remains unchanged by PrPC reduction after disease onset. These results define the potential of targeting PrPC as a disease-modifying therapy for certain AD-related phenotypes after disease onset. SIGNIFICANCE STATEMENT The study presented here further elucidates our understanding of the soluble oligomeric amyloid-β–Aβo-binding cellular prion protein (PrPC) signaling pathway in a familial form of Alzheimer's disease (AD) by implicating PrPC as a potential therapeutic target for AD. In particular, genetic deletion of Prnp rescued several familial AD (FAD)-associated phenotypes after disease onset in a mouse model of FAD. This study underscores the therapeutic potential of PrPC deletion given that patients already present symptoms at the time of diagnosis. PMID:28842420

Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

PubMed Central

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

2015-01-01

SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

Human Quiescin-sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis†

PubMed Central

Heckler, Erin J.; Alon, Assaf; Fass, Deborah; Thorpe, Colin

2013-01-01

The flavoprotein Quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active towards reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower kcat/Km for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70–C73) to a distal disulfide (C509–C512), then across the dimer interface to the FAD-proximal disulfide (C449–C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509–C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain. PMID:18393449

Elucidating nitric oxide synthase domain interactions by molecular dynamics.

PubMed

Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

2016-02-01

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis. © 2015 The Protein Society.

Insights into the Indian Peanut Genotypes for ahFAD2 Gene Polymorphism Regulating Its Oleic and Linoleic Acid Fluxes

PubMed Central

Nawade, Bhagwat; Bosamia, Tejas C.; Thankappan, Radhakrishnan; Rathnakumar, Arulthambi L.; Kumar, Abhay; Dobaria, Jentilal R.; Kundu, Rahul; Mishra, Gyan P.

2016-01-01

In peanut (Arachis hypogaea L.), the customization of fatty acid profile is an evolving area to fulfill the nutritional needs in the modern market. A total of 174 peanut genotypes, including 167 Indian cultivars, 6 advanced breeding lines and “SunOleic95R”—a double mutant line, were investigated using AS-PCRs, CAPS and gene sequencing for the ahFAD2 allele polymorphism, along with its fatty acid compositions. Of these, 80 genotypes were found having substitution (448G>A) mutation only in ahFAD2A gene, while none recorded 1-bp insertion (441_442insA) mutation in ahFAD2B gene. Moreover, 22 wild peanut accessions found lacking both the mutations. Among botanical types, the ahFAD2A mutation was more frequent in ssp. hypogaea (89%) than in ssp. fastigiata (17%). This single allele mutation, found affecting not only oleic to linoleic acid fluxes, but also the composition of other fatty acids in the genotypes studied. Repeated use of a few selected genotypes in the Indian varietal development programs were also eminently reflected in its ahFAD2 allele polymorphism. Absence of known mutations in the wild-relatives indicated the possible origin of these mutations, after the allotetraploidization of cultivated peanut. The SNP analysis of both ahFAD2A and ahFAD2B genes, revealed haplotype diversity of 1.05% and 0.95%, while Ka/Ks ratio of 0.36 and 0.39, respectively, indicating strong purifying selection pressure on these genes. Cluster analysis, using ahFAD2 gene SNPs, showed presence of both mutant and non-mutant genotypes in the same cluster, which might be due the presence of ahFAD2 gene families. This investigation provided insights into the large number of Indian peanut genotypes, covering various aspects related to O/L flux regulation and ahFAD2 gene polymorphism. PMID:27610115

Gene Expression of Desaturase (FADS1 and FADS2) and Elongase (ELOVL5) Enzymes in Peripheral Blood: Association with Polyunsaturated Fatty Acid Levels and Atopic Eczema in 4-Year-Old Children

PubMed Central

Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Àngels; López-Sabater, M. Carmen

2013-01-01

Abstract Background It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. Methods AE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson’s rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE. Results Children with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1. Conclusions and Significance Changes in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children. PMID:24167612

Gene expression of desaturase (FADS1 and FADS2) and Elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.

PubMed

Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Angels; López-Sabater, M Carmen

2013-01-01

It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. AE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson's rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE. Children with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1. Changes in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children.

Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library.

PubMed

Mu, Yi; Cai, Pengfei; Hu, Siqi; Ma, Sucan; Gao, Youhe

2014-01-01

Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

PubMed

Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Rowland, Gordon; Booker, Helen; You, Frank M; Cloutier, Sylvie

2013-10-01

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

Noise removal using factor analysis of dynamic structures: application to cardiac gated studies.

PubMed

Bruyant, P P; Sau, J; Mallet, J J

1999-10-01

Factor analysis of dynamic structures (FADS) facilitates the extraction of relevant data, usually with physiologic meaning, from a dynamic set of images. The result of this process is a set of factor images and curves plus some residual activity. The set of factor images and curves can be used to retrieve the original data with reduced noise using an inverse factor analysis process (iFADS). This improvement in image quality is expected because the inverse process does not use the residual activity, assumed to be made of noise. The goal of this work is to quantitate and assess the efficiency of this method on gated cardiac images. A computer simulation of a planar cardiac gated study was performed. The simulated images were added with noise and processed by the FADS-iFADS program. The signal-to-noise ratios (SNRs) were compared between original and processed data. Planar gated cardiac studies from 10 patients were tested. The data processed by FADS-iFADS were subtracted to the original data. The result of the substraction was studied to evaluate its noisy nature. The SNR is about five times greater after the FADS-iFADS process. The difference between original and processed data is noise only, i.e., processed data equals original data minus some white noise. The FADS-iFADS process is successful in the removal of an important part of the noise and therefore is a tool to improve the image quality of cardiac images. This tool does not decrease the spatial resolution (compared with smoothing filters) and does not lose details (compared with frequential filters). Once the number of factors is chosen, this method is not operator dependent.

Depressed expression of FAE1 and FAD2 genes modifies fatty acid profiles and storage compounds accumulation in Brassica napus seeds.

PubMed

Shi, Jianghua; Lang, Chunxiu; Wang, Fulin; Wu, Xuelong; Liu, Renhu; Zheng, Tao; Zhang, Dongqing; Chen, Jinqing; Wu, Guanting

2017-10-01

In plants, the enzymes fatty acid dehydrogenase 2 (FAD2) and fatty acid elongase 1 (FAE1) have been shown in previous studies to play important roles in the de novo biosynthesis of fatty acids. However, the effects of depressed expression of FAD2 and FAE1 on seed storage compounds accumulation remains to be elucidated. In this study, we produced RNA interfering transgenic rapeseeds lines, BnFAD2-Ri, BnFAE1-Ri and BnFAD2/BnFAE1-Ri, which exhibited depressed expression of the BnFAD2 and BnFAE1 genes under the control of seed-specific napin A promoter. These transgenic rapeseeds showed normal growth and development as compared with the wild type (CY2). Depressed expression of BnFAD2 and BnFAE1 genes modified fatty acid profiles, leading to increased oleic acid and decreased erucic acid contents in transgenic seeds. Consistent with these results, the ratios of C18:1/C18:2 and C18:1/C18:3 in C18 unsaturated fatty acids were greatly increased due to increased oleic acid content in transgenic seeds. Moreover, depressed expression of BnFAD2 and BnFAE1 genes resulted in slightly decreased oil contents and increased protein contents in transgenic seeds. Our results demonstrated that depressed expression of BnFAD2 and BnFAE1 greatly improves seed nutritional quality by modulating the fatty acid metabolism and storage products accumulation and that BnFAD2 and BnFAE1 are reliable targets for genetic improvement of rapeseed in seed nutritional quality. Copyright © 2017 Elsevier B.V. All rights reserved.

Cold perception and gene expression differ in Olea europaea seed coat and embryo during drupe cold acclimation.

PubMed

D'Angeli, S; Falasca, G; Matteucci, M; Altamura, M M

2013-01-01

FAD2 and FAD7 desaturases are involved in cold acclimation of olive (Olea europaea) mesocarp. There is no research information available on cold acclimation of seeds during mesocarp cold acclimation or on differences in the cold response of the seed coat and embryo. How FAD2 and FAD7 affect seed coat and embryo cold responses is unknown. Osmotin positively affects cold acclimation in olive tree vegetative organs, but its role in the seeds requires investigation. OeFAD2.1, OeFAD2.2, OeFAD7 and Oeosmotin were investigated before and after mesocarp acclimation by transcriptomic, lipidomic and immunolabelling analyses, and cytosolic calcium concentration ([Ca(2+)](cyt)) signalling, F-actin changes and seed development were investigated by epifluorescence/histological analyses. Transient [Ca(2+)](cyt) rises and F-actin disassembly were found in cold-shocked protoplasts from the seed coat, but not from the embryo. The thickness of the outer endosperm cuticle increased during drupe exposure to lowering of temperature, whereas the embryo protoderm always lacked cuticle. OeFAD2 transcription increased in both the embryo and seed coat in the cold-acclimated drupe, but linoleic acid (i.e. the product of FAD2 activity) increased solely in the seed coat. Osmotin was immunodetected in the seed coat and endosperm of the cold-acclimated drupe, and not in the embryo. The results show cold responsiveness in the seed coat and cold tolerance in the embryo. We propose a role for the seed coat in maintaining embryo cold tolerance by increasing endosperm cutinization through FAD2 and osmotin activities. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Fowl adenovirus-induced diseases and strategies for their control - a review on the current global situation.

PubMed

Schachner, Anna; Matos, Miguel; Grafl, Beatrice; Hess, Michael

2018-04-01

The stand-alone pathogenicity of fowl adenoviruses (FAdVs) had long been disputed, given the ubiquity of the viruses versus sporadic outbreaks, and variation between experimental studies. However, a globally emerging trend of FAdV-associated diseases has marked the past two decades, with hepatitis-hydropericardium syndrome mainly in Asia besides Arabian and Latin American countries, and geographically more disseminated outbreaks of inclusion body hepatitis. Finally, the appearance of FAdV-induced gizzard erosion (AGE) in Asia and Europe completed the range of diseases. Epidemiological studies confirmed serotype FAdV-4 as agent of hepatitis-hydropericardium syndrome, whereas inclusion body hepatitis is related to FAdV-2, -8a, -8b and -11. Members of the biologically more distant serotype FAdV-1 induce AGE. Urged by increasing problems in the field, numerous pathogenicity studies with FAdVs from outbreaks substantiated the primary aetiologic role of particular strains for distinct clinical conditions. Developments in the poultry industry towards highly specialized genetic breeds and rigorous biosecurity additionally contribute to the growing incidence of FAdV-related diseases. Confirming field observations, recent studies connected a higher susceptibility of broilers with their distinct physiology, implying the choice of bird type as a factor to be considered in infection studies. Furthermore, elevated biosecurity standards have generated immunologically naïve breeding stocks, putting broilers at risk in face of vertical FAdV transmission. Therefore, future prevention strategies should include adequate antibodies in breeders prior to production and - if necessary - vaccination, in order to protect progenies. This review aims to deliver a detailed overview on the current global situation about FAdV-induced diseases, their reproduction in vivo and vaccination strategies.

FAD286, an aldosterone synthase inhibitor, reduced atherosclerosis and inflammation in apolipoprotein E-deficient mice.

PubMed

Gamliel-Lazarovich, Aviva; Gantman, Anna; Coleman, Raymond; Jeng, Arco Y; Kaplan, Marielle; Keidar, Shlomo

2010-09-01

Aldosterone is known to be involved in atherosclerosis and cardiovascular disease and blockade of its receptor was shown to improve cardiovascular function. It was, therefore, hypothesized that inhibition of aldosterone synthesis would also reduce atherosclerosis development. To test this hypothesis, we examined the effect of FAD286 (FAD), an aldosterone synthase inhibitor, on the development of atherosclerosis in spontaneous atherosclerotic apolipoprotein E-deficient mice. Mice were divided into three treatment groups: normal diet, low-salt diet (LSD) and LSD treated with FAD at 30 mg/kg per day (LSD + FAD) for 10 weeks. Histomorphometry of the aortas obtained from these mice showed that atherosclerotic lesion area increased by three-fold under LSD compared with normal diet and FAD significantly reduced lesion area to values similar to normal diet. Changes in atherosclerosis were paralleled by changes in the expression of the inflammation markers (C-reactive protein, monocyte chemotactic protein-1, interleukin-6, nuclear factor kappa B and intercellular adhesion molecule-1) in peritoneal macrophages obtained from these mice. Surprisingly, whereas LSD increased serum or urine aldosterone levels, FAD did not alter these levels when evaluated at the end of the study. In J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, FAD was shown to have a direct dose-dependent anti-inflammatory effect. In apolipoprotein E-deficient mice, FAD reduces atherosclerosis and inflammation. However, these actions appeared to be dissociated from its effect on inhibition of aldosterone synthesis.

Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis*

PubMed Central

Le, Nguyen-Hung; Molle, Virginie; Eynard, Nathalie; Miras, Mathieu; Stella, Alexandre; Bardou, Fabienne; Galandrin, Ségolène; Guillet, Valérie; André-Leroux, Gwenaëlle; Bellinzoni, Marco; Alzari, Pedro; Mourey, Lionel; Burlet-Schiltz, Odile; Daffé, Mamadou; Marrakchi, Hedia

2016-01-01

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target. PMID:27590338

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

PubMed

Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

PubMed

Ogawara, Hiroshi

2016-09-01

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brizio, Carmen; Galluccio, Michele; Wait, Robin

2006-06-09

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-stepmore » affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.« less

Analysis and Results from a Flush Airdata Sensing System in Close Proximity to Firing Rocket Nozzles

NASA Technical Reports Server (NTRS)

Ali, Aliyah N.; Borrer, Jerry L.

2013-01-01

This paper presents information regarding the nosecap Flush Airdata Sensing (FADS) system on Orion’s Pad Abort 1 (PA-1) vehicle. The purpose of the nosecap FADS system was to test whether or not useful data could be obtained from a FADS system if it was placed in close proximity to firing rocket nozzles like the Attitude Control Motor (ACM) nozzles on the PA-1 Launch Abort System. The nosecap FADS system used pressure measurements from a series of pressure ports which were arranged in a cruciform pattern and flush with the surface of the vehicle to estimate values of angle of attack, angle of sideslip, Mach number, impact pressure, and freestream static pressure. This paper will present the algorithms employed by the FADS system along with the development of the calibration datasets and a comparison of the final results to the Best Estimated Trajectory (BET) data for PA-1. Also presented in this paper is a Computational Fluid Dynamics (CFD) study to explore the impact of the ACM on the nosecap FADS system. The comparison of the nosecap FADS system results to the BET and the CFD study showed that more investigation is needed to quantify the impact of the firing rocket motors on the FADS system.

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins.

PubMed

Sun, D; Leung, C L; Liem, R K

2001-01-01

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3' end of the MACF cDNA; hence, we refer to it as MACF2. The C-terminal domain of mouse MACF2 was cloned and characterized. The microtubule-binding properties of MACF2 C-terminal domain are similar to that of MACF. The GAR domain was originally found in Gas 2 protein and here we show that it can associate with MTs in transfected cells. Plectin and desmoplakin have GSR-containing domains at their C-termini and we further demonstrate that the GSR-containing domain of plectin, but not desmoplakin, can bind to MTs in vivo.

Performance of rumpon-based tuna fishery in the Fishing Port of Sendangbiru, Malang, Indonesia

NASA Astrophysics Data System (ADS)

Wiadnya, D. G. R.; Damora, A.; Tamanyira, M. M.; Nugroho, D.; Darmawan, A.

2018-03-01

Catch records on FADs-based tuna fishery (hand-line) in the southern of East Java was conducted in fishing base of Sendangbiru, from February 2016 to May 2017. Total 45 rumpon (FADs) were found within space area of 64,081 square nautical miles, with average between FADs distant of 25 nautical miles. All FADs were located outside Fisheries Management Areas, except one FAD of code 31. Hand-line fishery was designed for a maximum fishing trip of 14 days. Total catch biomass mainly determined fishing trip. When catch exceeded 1,000 kg, fishermen tend to turn back although with < 10 days fishing trip. Yellowfin tuna and skipjacks were two main species that formed total catch with average catch biomass varied amongst 425–1,360 kg trip-1. Apart from baby-tuna, yellowfin tuna and albacore within catch were at size class higher than its first maturity stage. However, length-class of skipjack indicated that it still in immature stage. Despite of FAD license, Ministry of Marine Affairs and Fisheries neither informed nor aware the position of these FADs.

Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site.

PubMed

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R; Kenniston, Jon A; Mendrola, Jeannine M; Ferguson, Kathryn M; Lemmon, Mark A

2015-02-03

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

DOE PAGES

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; ...

2015-01-22

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here in this paper, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound tomore » an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. In conclusion, our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.« less

Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heroux, A.; Bozinovski, D; Valley, M

2009-01-01

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. Themore » oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.« less

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.

2012-03-15

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure ofmore » human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.« less

Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA*

PubMed Central

Sharma, Amit; Jenkins, Katherine R.; Héroux, Annie; Bowman, Gregory D.

2011-01-01

Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves. PMID:22033927

Multiple Animal Studies for Medical Chemical Defense Program in Soldier/ Patient Decontamination and Drug Development on Task Order 84-6: Pyruvate Dehydrogenase System for Determining the Effectiveness of Arsenic Antidotes

DTIC Science & Technology

1988-03-11

adenine dinucleotide FAD = flavin-adenine dinucleotide iipS2 = lipoic acid lip(SH)2 = dihydrolipoic acid CoA = coenzyme A. SHepatic PDH complex activity...tissues has yet to be fully characterized, but it probably involves arsenic binding to the lipoic acid and dithiol moieties of the complex (Fluharty...covalently bound lipoic acid substrate of dihydrolipoyl transacetylase is greater per mole of L and CVAA than for sodium arsenite. This is possible

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calvo, Eric; Mans, Ben J.; Ribeiro, José M.C.

The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describemore » the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.« less

FadD Is Required for Utilization of Endogenous Fatty Acids Released from Membrane Lipids ▿ †

PubMed Central

Pech-Canul, Ángel; Nogales, Joaquina; Miranda-Molina, Alfonso; Álvarez, Laura; Geiger, Otto; Soto, María José; López-Lara, Isabel M.

2011-01-01

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth. PMID:21926226

Multidomain flavin-dependent sulfhydryl oxidases.

PubMed

Coppock, Donald L; Thorpe, Colin

2006-01-01

Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand-alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs.

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

PubMed Central

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

A confirmative clinimetric analysis of the 36-item Family Assessment Device.

PubMed

Timmerby, Nina; Cosci, Fiammetta; Watson, Maggie; Csillag, Claudio; Schmitt, Florence; Steck, Barbara; Bech, Per; Thastum, Mikael

2018-02-07

The Family Assessment Device (FAD) is a 60-item questionnaire widely used to evaluate self-reported family functioning. However, the factor structure as well as the number of items has been questioned. A shorter and more user-friendly version of the original FAD-scale, the 36-item FAD, has therefore previously been proposed, based on findings in a nonclinical population of adults. We aimed in this study to evaluate the brief 36-item version of the FAD in a clinical population. Data from a European multinational study, examining factors associated with levels of family functioning in adult cancer patients' families, were used. Both healthy and ill parents completed the 60-item version FAD. The psychometric analyses conducted were Principal Component Analysis and Mokken-analysis. A total of 564 participants were included. Based on the psychometric analysis we confirmed that the 36-item version of the FAD has robust psychometric properties and can be used in clinical populations. The present analysis confirmed that the 36-item version of the FAD (18 items assessing 'well-being' and 18 items assessing 'dysfunctional' family function) is a brief scale where the summed total score is a valid measure of the dimensions of family functioning. This shorter version of the FAD is, in accordance with the concept of 'measurement-based care', an easy to use scale that could be considered when the aim is to evaluate self-reported family functioning.

Factor for adipocyte differentiation 158 gene disruption prevents the body weight gain and insulin resistance induced by a high-fat diet.

PubMed

Hayashi, Takahiro; Nozaki, Yuriko; Nishizuka, Makoto; Ikawa, Masahito; Osada, Shigehiro; Imagawa, Masayoshi

2011-01-01

To clarify the molecular mechanism of adipocyte differentiation, we previously isolated a novel gene, factor for adipocyte differentiation (fad) 158, whose expression was induced during the earliest stages of adipogenesis, and its product was localized to the endoplasmic reticulum. We found that the knockdown of fad158 expression prevented the differentiation of 3T3-L1 cells into adipocytes. In addition, over-expression of fad158 promoted the differentiation of NIH-3T3 cells, which do not usually differentiate into adipocytes. Although these findings strongly suggest that fad158 has a crucial role in regulating adipocyte differentiation, the physiological role of the gene is still unclear. In this study, we generated mice in which fad158 expression was deleted. The fad158-deficient mice did not show remarkable changes in body weight or the weight of white adipose tissue on a chow diet, but had significantly lower body weights and fat mass than wild-type mice when fed a high-fat diet. Furthermore, although the disruption of fad158 did not influence insulin sensitivity on the chow diet, it improved insulin resistance induced by the high-fat diet. These results indicate that fad158 is a key factor in the development of obesity and insulin resistance caused by a high-fat diet.

Fatty acid desaturase 1 knockout mice are lean with improved glycemic control and decreased development of atheromatous plaque

PubMed Central

Powell, David R; Gay, Jason P; Smith, Melinda; Wilganowski, Nathaniel; Harris, Angela; Holland, Autumn; Reyes, Maricela; Kirkham, Laura; Kirkpatrick, Laura L; Zambrowicz, Brian; Hansen, Gwenn; Platt, Kenneth A; van Sligtenhorst, Isaac; Ding, Zhi-Ming; Desai, Urvi

2016-01-01

Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for ω3, ω6, and ω9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. PMID:27382320

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

PubMed

Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

2016-04-07

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Ligand Binding to WW Tandem Domains of YAP2 Transcriptional Regulator Is Under Negative Cooperativity

PubMed Central

Schuchardt, Brett J.; Mikles, David C.; Hoang, Lawrence M.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

2014-01-01

YAP2 transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well-documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. PMID:25283809

Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE).

PubMed

Buyannemekh, Dolgorsuren; Nham, Sang-Uk

2017-05-31

The β2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of β2 integrin, αMβ2 and αXβ2, share the leukocyte distribution profile and integrin αXβ2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and αXβ2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of αXβ2, we characterize the binding nature and the interacting moieties of αX I-domain and RAGE. Their binding requires divalent cations (Mg 2+ and Mn 2+ ) and shows an affinity on the sub-micro molar level: the dissociation constant of αX I-domains binding to RAGE being 0.49 μM. Furthermore, the αX I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of αX I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to αX I-domain. In conclusion, the main mechanism of αX I-domain binding to RAGE is a charge interaction, in which the acidic moieties of αX I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Src binds cortactin through an SH2 domain cystine-mediated linkage.

PubMed

Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

2012-12-15

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

Src binds cortactin through an SH2 domain cystine-mediated linkage

PubMed Central

Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

2012-01-01

Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

PubMed

Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

2006-12-01

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

FADS1 genetic variability interacts with dietary α-linolenic acid intake to affect serum non-HDL-cholesterol concentrations in European adolescents.

PubMed

Dumont, Julie; Huybrechts, Inge; Spinneker, Andre; Gottrand, Frédéric; Grammatikaki, Evangelia; Bevilacqua, Noemi; Vyncke, Krishna; Widhalm, Kurt; Kafatos, Anthony; Molnar, Denes; Labayen, Idoia; Gonzalez-Gross, Marcela; Amouyel, Philippe; Moreno, Luis A; Meirhaeghe, Aline; Dallongeville, Jean

2011-07-01

Two rate-limiting enzymes in PUFA biosynthesis, Δ5- and Δ6-desaturases, are encoded by the FADS1 and FADS2 genes, respectively. Genetic variants in the FADS1-FADS2 gene cluster are associated with changes in plasma concentrations of PUFA, HDL- and LDL-cholesterol, and TG. However, little is known about whether dietary PUFA intake modulates these associations, especially in adolescents. We assessed whether dietary linoleic acid (LA) or α-linolenic acid (ALA) modulate the association between the FADS1 rs174546 polymorphism and concentrations of PUFA, other lipids, and lipoproteins in adolescents. Dietary intakes of LA and ALA, FADS1 rs174546 genotypes, PUFA levels in serum phospholipids, and serum concentrations of TG, cholesterol, and lipoproteins were determined in 573 European adolescents from the HELENA study. The sample was stratified according to the median dietary LA (≤9.4 and >9.4 g/d) and ALA (≤1.4 and >1.4 g/d) intakes. The associations between FADS1 rs174546 and concentrations of PUFA, TG, cholesterol, and lipoproteins were not affected by dietary LA intake (all P-interaction > 0.05). Similarly, the association between the FADS1 rs174546 polymorphism and serum phospholipid concentrations of ALA or EPA was not modified by dietary ALA intake (all P-interaction > 0.05). In contrast, the rs174546 minor allele was associated with lower total cholesterol concentrations (P = 0.01 under the dominant model) and non-HDL-cholesterol concentrations (P = 0.02 under the dominant model) in the high-ALA-intake group but not in the low-ALA-intake group (P-interaction = 0.01). These results suggest that dietary ALA intake modulates the association between FADS1 rs174546 and serum total and non-HDL-cholesterol concentrations at a young age.

1950 MHz Electromagnetic Fields Ameliorate Aβ Pathology in Alzheimer’s Disease Mice

PubMed Central

Jeong, Ye Ji; Kang, Ga-Young; Kwon, Jong Hwa; Choi, Hyung-Do; Pack, Jeong-Ki; Kim, Nam; Lee, Yun-Sil; Lee, Hae-June

2015-01-01

The involvement of radiofrequency electromagnetic fields (RF-EMF) in the neurodegenerative disease, especially Alzheimer’s disease (AD), has received wide consideration, however, outcomes from several researches have not shown consistency. In this study, we determined whether RF-EMF influenced AD pathology in vivo using Tg-5xFAD mice as a model of AD-like amyloid β (Aβ) pathology. The transgenic (Tg)-5xFAD and wild type (WT) mice were chronically exposed to RF-EMF for 8 months (1950 MHz, SAR 5W/kg, 2 hrs/day, 5 days/week). Notably, chronic RF-EMF exposure significantly reduced not only Aβ plaques, APP, and APP carboxyl-terminal fragments (CTFs) in whole brain including hippocampus and entorhinal cortex but also the ratio of Aβ42 and Aβ40 peptide in the hippocampus of Tg-5xFAD mice. We also found that parenchymal expression of β-amyloid precursor protein cleaving enzyme 1(BACE1) and neuroinflammation were inhibited by RF-EMF exposure in Tg-5xFAD. In addition, RF-EMF was shown to rescue memory impairment in Tg-5xFAD. Moreover, gene profiling from microarray data using hippocampus of WT and Tg-5xFAD following RF-EMF exposure revealed that 5 genes (Tshz2, Gm12695, St3gal1, Isx and Tll1), which are involved in Aβ, are significantly altered inTg-5xFAD mice, exhibiting different responses to RF-EMF in WT or Tg-5xFAD mice; RF-EMF exposure in WT mice showed similar patterns to control Tg-5xFAD mice, however, RF-EMF exposure in Tg-5xFAD mice showed opposite expression patterns. These findings indicate that chronic RF-EMF exposure directly affects Aβ pathology in AD but not in normal brain. Therefore, RF-EMF has preventive effects against AD-like pathology in advanced AD mice with a high expression of Aβ, which suggests that RF-EMF can have a beneficial influence on AD. PMID:26017559

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains.

PubMed

Zhou, Huan-Xiang

2006-11-01

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin

PubMed Central

Rooijakkers, Bart J. M.

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain’s sequence–function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed. PMID:29782536

Design and Calibration of the X-33 Flush Airdata Sensing (FADS) System

NASA Technical Reports Server (NTRS)

Whitmore, Stephen A.; Cobleigh, Brent R.; Haering, Edward A.

1998-01-01

This paper presents the design of the X-33 Flush Airdata Sensing (FADS) system. The X-33 FADS uses a matrix of pressure orifices on the vehicle nose to estimate airdata parameters. The system is designed with dual-redundant measurement hardware, which produces two independent measurement paths. Airdata parameters that correspond to the measurement path with the minimum fit error are selected as the output values. This method enables a single sensor failure to occur with minimal degrading of the system performance. The paper shows the X-33 FADS architecture, derives the estimating algorithms, and demonstrates a mathematical analysis of the FADS system stability. Preliminary aerodynamic calibrations are also presented here. The calibration parameters, the position error coefficient (epsilon), and flow correction terms for the angle of attack (delta alpha), and angle of sideslip (delta beta) are derived from wind tunnel data. Statistical accuracy of' the calibration is evaluated by comparing the wind tunnel reference conditions to the airdata parameters estimated. This comparison is accomplished by applying the calibrated FADS algorithm to the sensed wind tunnel pressures. When the resulting accuracy estimates are compared to accuracy requirements for the X-33 airdata, the FADS system meets these requirements.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

PubMed

Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP).

PubMed

Lessard, Christian B; Cottrell, Barbara A; Maruyama, Hiroko; Suresh, Suraj; Golde, Todd E; Koo, Edward H

2015-01-01

The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj; Shaghasi, Tarana

The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj; Shagasi, Tarana

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj; Shagasi, Tarana

2015-06-30

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stringer, Mary Ann; McBrayer, Brett

2016-11-29

The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

Structure of the human sulfhydryl oxidase augmenter of liver regeneration and characterization of a human mutation causing an autosomal recessive myopathy†, ‡

PubMed Central

Daithankar, Vidyadhar N.; Schaefer, Stephanie A.; Dong, Ming; Bahnson, Brian J.; Thorpe, Colin

2010-01-01

The sulfhydryl oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short, cytokine-like, form of ALR (sfALR), and a longer form (lfALR) which resides in the mitochondrial intermembrane space (IMS). lfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia40 during oxidative protein folding within the IMS. Recently Di Fonzo et al. (Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G. P. (2009) Am. J. Hum. Genet. 84, 594–604) described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human R194H mutant in lf- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR, close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself, and from C95 of the intersubunit disulfide of the other protomer) and with the 2' OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However the mutation adversely affects the stability of both ALR forms: e.g. by decreasing the melting temperature by about 10 °C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NMR spectra of wild type sfALR and its R194H mutant reveal a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al. PMID:20593814

A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding.

PubMed

Stowell, James A W; Wagstaff, Jane L; Hill, Chris H; Yu, Minmin; McLaughlin, Stephen H; Freund, Stefan M V; Passmore, Lori A

2018-06-15

Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1-RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding. © 2018 Stowell et al.

Measuring family functioning in families with parental cancer: Reliability and validity of the German adaptation of the Family Assessment Device (FAD).

PubMed

Beierlein, Volker; Bultmann, Johanna Christine; Möller, Birgit; von Klitzing, Kai; Flechtner, Hans-Henning; Resch, Franz; Herzog, Wolfgang; Brähler, Elmar; Führer, Daniel; Romer, Georg; Koch, Uwe; Bergelt, Corinna

2017-02-01

The concept of family functioning is gaining importance in psycho-oncology research and health care services. The Family Assessment Device (FAD) is a well-established measure of family functioning. Psychometric properties inherent in the German 51-item adaptation of the FAD are examined in different samples of families with parental cancer. Acceptance, reliability, and validity of FAD scales are analysed in samples from different study settings (N=1701 cancer patients, N=261 partners, N=158 dependent adolescent children 11 to 18years old). Missing items in the FAD scales (acceptance) are rare for adults (<1.1%) and adolescent children (<4.4%). In samples of adults and older adolescents (15 to 18years), all FAD scales except for the Roles scale are significantly reliable (0.75≤Cronbach's α≤0.88). The scales correlate highly (0.46≤Pearson's r≤0.59) with the criterion satisfaction with family life (convergent validity), and have smaller correlations (0.16≤r≤0.49) with measures of emotional distress and subjective well-being (divergent validity). In most FAD scales, adults seeking family counselling report worse family functioning (0.24≤Cohen's d≤0.59) than adults in other samples with parental cancer (discriminative validity). Overall, the German 51-item adaptation of the FAD reveals good acceptance, reliability, and validity for cancer patients and their relatives. Particularly the scale General Functioning shows excellent psychometric properties. The FAD is suitable in the assessment of families with parental cancer for adults and adolescents older than 11years. Copyright © 2016 Elsevier Inc. All rights reserved.

An Amino Acids Mixture Attenuates Glycemic Impairment but not Affects Adiposity Development in Rats Fed with AGEs-containing Diet

PubMed Central

Liao, Yi-Hung; Chen, Chung-Yu; Chen, Chiao-Nan; Wu, Chia-Ying; Tsai, Shiow-Chwen

2018-01-01

Background: Unhealthy western dietary patterns lead to over-consumption of fat and advanced glycation end-products (AGEs), and these account for the developments of obesity, diabetes, and related metabolic disorders. Certain amino acids (AAs) have been recently demonstrated to improve glycemia and reduce adiposity. Therefore, our primary aims were to examine whether feeding an isoleucine-enriched AA mixture (4.5% AAs; Ile: 3.0%, Leu: 1.0%, Val: 0.2%, Arg: 0.3% in the drinking water) would affect adiposity development and prevent the impairments of glycemic control in rats fed with the fat/AGE-containing diet (FAD). Methods: Twenty-four male Sprague-Dawley rats were assigned into 1) control diet (CD, N = 8), 2) FAD diet (FAD, N = 8), and 3) FAD diet plus AA (FAD/AA, N = 8). After 9-weeks intervention, the glycemic control capacity (glucose level, ITT, and HbA1c levels), body composition, and spontaneous locomotor activity (SLA) were evaluated, and the fasting blood samples were collected for analyzing metabolic related hormones (insulin, leptin, adiponectin, and corticosterone). The adipose tissues were also surgically collected and weighed. Results: FAD rats showed significant increases in weight gain, body fat %, blood glucose, HbA1c, leptin, and area under the curve of glucose during insulin tolerance test (ITT-glucose-AUC) in compared with the CD rats. However, the fasting levels of blood glucose, HbA1c, leptin, and ITT-glucose-AUC did not differ between CD and FAD/AA rats. FAD/AA rats also showed a greater increase in serum testosterone. Conclusion: The amino acid mixture consisting of Ile, Leu, Val, and Arg showed clear protective benefits on preventing the FAD-induced obesity and impaired glycemic control. PMID:29333102

An Amino Acids Mixture Attenuates Glycemic Impairment but not Affects Adiposity Development in Rats Fed with AGEs-containing Diet.

PubMed

Liao, Yi-Hung; Chen, Chung-Yu; Chen, Chiao-Nan; Wu, Chia-Ying; Tsai, Shiow-Chwen

2018-01-01

Background: Unhealthy western dietary patterns lead to over-consumption of fat and advanced glycation end-products (AGEs), and these account for the developments of obesity, diabetes, and related metabolic disorders. Certain amino acids (AAs) have been recently demonstrated to improve glycemia and reduce adiposity. Therefore, our primary aims were to examine whether feeding an isoleucine-enriched AA mixture (4.5% AAs; Ile: 3.0%, Leu: 1.0%, Val: 0.2%, Arg: 0.3% in the drinking water) would affect adiposity development and prevent the impairments of glycemic control in rats fed with the fat/AGE-containing diet (FAD). Methods: Twenty-four male Sprague-Dawley rats were assigned into 1) control diet (CD, N = 8), 2) FAD diet (FAD, N = 8), and 3) FAD diet plus AA (FAD/AA, N = 8). After 9-weeks intervention, the glycemic control capacity (glucose level, ITT, and HbA1c levels), body composition, and spontaneous locomotor activity (SLA) were evaluated, and the fasting blood samples were collected for analyzing metabolic related hormones (insulin, leptin, adiponectin, and corticosterone). The adipose tissues were also surgically collected and weighed. Results: FAD rats showed significant increases in weight gain, body fat %, blood glucose, HbA1c, leptin, and area under the curve of glucose during insulin tolerance test (ITT-glucose-AUC) in compared with the CD rats. However, the fasting levels of blood glucose, HbA1c, leptin, and ITT-glucose-AUC did not differ between CD and FAD/AA rats. FAD/AA rats also showed a greater increase in serum testosterone. Conclusion: The amino acid mixture consisting of Ile, Leu, Val, and Arg showed clear protective benefits on preventing the FAD-induced obesity and impaired glycemic control.

Application of cross-priming amplification (CPA) for detection of fowl adenovirus (FAdV) strains.

PubMed

Niczyporuk, Jowita Samanta; Woźniakowski, Grzegorz; Samorek-Salamonowicz, Elżbieta

2015-04-01

Fowl adenoviruses (FAdVs) are widely distributed among chickens. Detection of FAdVs is mainly accomplished by virus isolation, serological assays, various polymerase chain reaction (PCR) assays, and loop-mediated isothermal amplification (LAMP). To increase the diagnostic capacity of currently applied techniques, cross-priming amplification (CPA) for the detection of the FAdV hexon gene was developed. The single CPA assay was optimised to detect all serotypes 1-8a-8b-11 representing the species Fowl aviadenovirus A-E. The optimal temperature and incubation time were determined to be 68 °C for 2 h. Using different incubation temperatures, it was possible to differentiate some FAdV serotypes. The results were recorded after addition of SYBR Green I(®) dye, which produced a greenish fluorescence under UV light. The CPA products separated by gel electrophoresis showed different "ladder-like" patterns for the different serotypes. The assay was specific for all serotypes of FAdV, and no cross-reactivity was observed with members of the genus Atadenovirus, duck atadenovirus A (egg drop syndrome virus EDS-76 [EDSV]) or control samples containing Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV) or chicken anaemia virus (CAV). The results of the newly developed FAdV-CPA were compared with those of real-time PCR. The sensitivity of CPA was equal to that of real-time PCR and reached 10(-2.0) TCID50, but the CPA method was more rapid and cheaper than the PCR systems. CPA is a highly specific, sensitive, efficient, and rapid tool for detection of all FAdV serotypes. This is the first report on the application of CPA for detection of FAdV strains.

I Can Stomach That! Fearlessness About Death Predicts Attenuated Facial Electromyography Activity in Response to Death-Related Images.

PubMed

Velkoff, Elizabeth A; Forrest, Lauren N; Dodd, Dorian R; Smith, April R

2016-06-01

Objective measures of suicide risk can convey life-saving information to clinicians, but few such measures exist. This study examined an objective measure of fearlessness about death (FAD), testing whether FAD relates to self-reported and physiological aversion to death. Females (n = 87) reported FAD and disgust sensitivity, and facial electromyography was used to measure physiological facial responses consistent with disgust while viewing death-related images. FAD predicted attenuated expression of physiological death aversion, even when controlling for self-reported death-related disgust sensitivity. Diminished physiological aversion to death-related stimuli holds promise as an objective measure of FAD and suicide risk. © 2015 The American Association of Suicidology.

Postpartum changes in maternal and infant erythrocyte fatty acids are likely to be driven by restoring insulin sensitivity and DHA status.

PubMed

Kuipers, Remko S; Luxwolda, Martine F; Sango, Wicklif S; Kwesigabo, Gideon; Velzing-Aarts, Francien V; Dijck-Brouwer, D A Janneke; Muskiet, Frits A J

2011-06-01

Perinatal changes in maternal glucose and lipid fluxes and de novo lipogenesis (DNL) are driven by hormones and nutrients. Docosahexaenoic acid (DHA) reduces, whereas insulin augments, nuclear abundance of sterol-regulatory-element-binding-protein-1 (SREBP-1), which promotes DNL, stearoyl-CoA-desaturase (SCD, also Δ9-desaturase), fatty acid-(FA)-elongation (Elovl) and FA-desaturation (FADS). Decreasing maternal insulin sensitivity with advancing gestation and compensatory hyperinsulinemia cause augmented postprandial glucose levels, adipose tissue lipolysis and hepatic glucose- and VLDL-production. Hepatic VLDL is composed of dietary, body store and DNL derived FA. Decreasing insulin sensitivity increases the contribution of FA from hepatic-DNL in VLDL-triacylglycerols, and consequently saturated-FA and monounsaturated-FA (MUFA) in maternal serum lipids increase during pregnancy. Although other authors described changes in maternal serum and RBC essential-FA (EFA) after delivery, none went into detail about the changes in non-EFA and the mechanisms behind -and/or functions of- the observed changes. Postpartum FA-changes result from changing enzymatic activities that are influenced by the changing hormonal milieu after delivery and DHA-status. We studied FA-profiles and FA-ratios (as indices for enzymatic activities) of maternal and infant RBC at delivery and after 3 months exclusive breastfeeding in three populations with increasing freshwater-fish intakes. DNL-, SCD- and FADS2-activities decreased after delivery. Elongation-6 (Elovl-6)- and FADS1-activities increased. The most pronounced postpartum changes for mothers were increases in 18:0, linoleic (LA), arachidonic acid (AA) and decreases in 16:0, 18:1ω9 and DHA; and for infants increases in 18:1ω9, 22:5ω3, LA and decreases in 16:0 and AA. Changes were in line with the literature. Postpartum increases in 18:0, and decreases in 16:0 and 18:1ω9, might derive from reduced insulin-promoted DNL-activity, with more reduced SCD- than Elovl-activity that leaves more 16:0 to be converted to 18:0 (Elovl-activity) than to MUFA (SCD-activity). Postpartum changes in ΣDNL, saturated-FA and MUFA related negatively to RBC-DHA. This concurs with suppression of both SCD- and Elovl-6 activities by DHA, through its influence on SREBP. Infant MUFA and LA increased at expense of their mothers. Sustained transport might be important for myelination (MUFA) and skin barrier development (LA). Maternal postpartum decreases in FADS2-, and apparent increases in FADS1-activity, together with increases in LA, AA, and 22:5ω3, but decrease in DHA, confirm that FADS2 is rate limiting in EFA-desaturation. Maternal LA and AA increases might be the result of rerouting from transplacental transfer to the incorporation into milk lipids and discontinued placental AA-utilization. Perinatal changes in maternal and infant FA status may be strongly driven by changing insulin sensitivity and DHA status. Copyright © 2011 Elsevier Ltd. All rights reserved.

Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

PubMed

Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

2017-07-25

Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

Identification and characterization of a plastidial ω-3 fatty acid desaturase EgFAD8 from oil palm (Elaeis guineensis Jacq.) and its promoter response to light and low temperature

PubMed Central

Chen, Lizhi; Wang, Lei; Wang, Herong; Sun, Ruhao; You, Lili; Zheng, Yusheng; Yuan, Yijun

2018-01-01

In higher plants, ω-3 fatty acid desaturases are the key enzymes in the biosynthesis of alpha-linolenic acid (18:3), which plays key roles in plant metabolism as a structural component of both storage and membrane lipids. Here, the first ω-3 fatty acid desaturase gene was identified and characterized from oil palm. The bioinformatic analysis indicated it encodes a temperature-sensitive chloroplast ω-3 fatty acid desaturase, designated as EgFAD8. The expression analysis revealed that EgFAD8 is highly expressed in the oil palm leaves, when compared with the expression in the mesocarp. The heterologous expression of EgFAD8 in yeast resulted in the production of a novel fatty acid 18:3 (about 0.27%), when fed with 18:2 in the induction culture. Furthermore, to detect whether EgFAD8 could be induced by the environment stress, we detected the expression efficiency of the EgFAD8 promoter in transgenic Arabidopsis treated with low temperature and darkness, respectively. The results indicated that the promoter of EgFAD8 gene could be significantly induced by low temperature and slightly induced by darkness. These results reveal the function of EgFAD8 and the feature of its promoter from oil palm fruits, which will be useful for understanding the fuction and regulation of plastidial ω-3 fatty acid desaturases in higher plants. PMID:29698515

Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Xu, Minghua; Wu, Nan

2006-12-01

The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6{sub 1} or P6{sub 5}, and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: themore » intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.« less

A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.

PubMed

Essen, L O; Perisic, O; Lynch, D E; Katan, M; Williams, R L

1997-03-11

We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

PubMed

Schuchardt, Brett J; Mikles, David C; Hoang, Lawrence M; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

2014-12-01

YES-associated protein 2 (YAP2) transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of the WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. © 2014 FEBS.

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

PubMed Central

Pérez, Yolanda; Maffei, Mariano; Igea, Ana; Amata, Irene; Gairí, Margarida; Nebreda, Angel R.; Bernadó, Pau; Pons, Miquel

2013-01-01

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation. PMID:23416516

GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

PubMed Central

Marino, Michael; Banerjee, Manidipa; Jonquières, Renaud; Cossart, Pascale; Ghosh, Partho

2002-01-01

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion. PMID:12411480

Genetic Variants of the FADS Gene Cluster Are Associated with Erythrocyte Membrane LC PUFA Levels in Patients with Mild Cognitive Impairment.

PubMed

Schuchardt, J P; Köbe, T; Witte, V; Willers, J; Gingrich, A; Tesky, V; Pantel, J; Rujescu, D; Illig, T; Flöel, A; Hahn, A

2016-01-01

Long-chain (> 20 C-atoms) polyunsaturated fatty acids (LC PUFAs) of both the omega-6 (n-6) and omega-3 (n-3) series are important for the functional integrity of brain and thereby cognition, memory and mood. Clinical studies observed associations between altered LC PUFA levels and neurodegenerative diseases such as Alzheimer´s disease and its prodromal stage, mild cognitive impairment (MCI). The present study examined the LC PUFA status of MCI patients with specific view on the relative LC n-3 PUFA levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in erythrocyte membranes (omega-3 index). 12 single nucleotide polymorphisms (SNPs) of the FADS1, FADS2, and FADS3 gene clusters were genotyped in 111 MCI patients and evaluated associations with PUFA levels in erythrocyte membranes (primary outcome). In addition, the associations between FADS SNPs and LC PUFA levels with serum lipid levels as well as depressive symptoms were examined (secondary outcomes). Minor allele carrier of rs174546, rs174548 (FADS1), rs3834458, rs1535, rs174574, rs174575, rs174576, and rs174578 (FADS2) showed significant higher n-6 and n-3 precursor PUFA levels (linoleic acid, and alpha-linolenic acid, respectively) and lower arachidonic acid (AA) levels in erythrocyte membranes compared to the major allele carriers. Differences in EPA and DHA levels were not significant. Minor allele carriers of rs174574, rs174576 and rs174578 (FADS2) and rs174455 (FADS3) exhibited significant higher triglyceride levels, whereas minor allele carriers for rs174449 and rs174455 (FADS3) exhibited significant higher total- and LDL-cholesterol levels compared to the more common variant. The mean omega-3 index of the study cohort was 6.19 ± 1.55 %. In more than 85 % of the patients, the omega-3 index was below 8 % and in 23 % below 5 %. Moreover, it was shown that a low DHA status and omega-3 index was associated with depressive symptoms (Beck's depression-inventory). These findings indicate an association between several FADS genotypes for higher n-6 and n-3 precursor PUFA and lower AA levels in erythrocyte membranes in minor compared to major allele carriers. To what extent FADS genotypes and a lower conversion of LA and ALA to biologically important LC PUFAs such as AA, EPA and DHA contributes to cognitive decline should be investigated in further trials. Nevertheless, the omega-3 index in this cohort of MCI patients can be classified as insufficient.

The HhH(2)/NDD Domain of the Drosophila Nod Chromokinesin-like Protein Is Required for Binding to Chromosomes in the Oocyte Nucleus

PubMed Central

Cui, Wei; Hawley, R. Scott

2005-01-01

Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9–10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes. PMID:16143607

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4*

PubMed Central

Biswas-Fiss, Esther E.; Affet, Stephanie; Ha, Malissa; Biswas, Subhasis B.

2012-01-01

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. PMID:23144455

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

2015-08-21

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association withmore » the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.« less

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

PubMed

Ono, S; Hirano, H

1984-04-01

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

Analysis and Results from a Flush Airdata Sensing (FADS) System in Close Proximity to Firing Rocket Nozzles

NASA Technical Reports Server (NTRS)

Ali, Aliyah N.; Borrer, Jerry L.

2013-01-01

This presentation presents information regarding the nose-cap flush airdata sensing (FADS) system on Orion's Pad Abort 1 (PA-1) vehicle. The purpose of the nose-cap FADS system was to test whether or not useful data could be obtained from a FADS system if it was placed in close proximity to firing rockets nozzles like the attitude control motor (ACM) nozzles on the PA-1 launch abort system (LAS). The nose-cap FADS systems use pressure measurements from a series of pressure ports which are arranged in a cruciform pattern and flush with the surface of the vehicle to estimate values of angle of attack, angle of side-slip, Mach number, impact pressure and free-stream static pressure.

Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors.

PubMed

Tsuruoka, Nozomu; Sadakane, Takuya; Hayashi, Rika; Tsujimura, Seiya

2017-03-10

The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus species require suitable redox mediators to transfer electrons from the enzyme to the electrode surface for the application of bioelectrical devices. Although several mediators for FAD-GDH are already in use, they are still far from optimum in view of potential, kinetics, sustainability, and cost-effectiveness. Herein, we investigated the efficiency of various phenothiazines and quinones in the electrochemical oxidation of FAD-GDH from Aspergillus terreus . At pH 7.0, the logarithm of the bimolecular oxidation rate constants appeared to depend on the redox potentials of all the mediators tested. Notably, the rate constant of each molecule for FAD-GDH was approximately 2.5 orders of magnitude higher than that for glucose oxidase from Aspergillus sp. The results suggest that the electron transfer kinetics is mainly determined by the formal potential of the mediator, the driving force of electron transfer, and the electron transfer distance between the redox active site of the mediator and the FAD, affected by the steric or chemical interactions. Higher k ₂ values were found for ortho-quinones than for para-quinones in the reactions with FAD-GDH and glucose oxidase, which was likely due to less steric hindrance in the active site in the case of the ortho-quinones.

The PpaA/AerR regulators of photosynthesis gene expression from anoxygenic phototrophic proteobacteria contain heme-binding SCHIC domains.

PubMed

Moskvin, Oleg V; Gilles-Gonzalez, Marie-Alda; Gomelsky, Mark

2010-10-01

The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

PubMed

Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

1998-11-10

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition

PubMed Central

Jun, Susie; Wallen, Robert V.; Goriely, Anne; Kalionis, Bill; Desplan, Claude

1998-01-01

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix–turn–helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes. PMID:9811867

Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

PubMed Central

Pan, Di; Song, Yuhua

2010-01-01

Abstract N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities. PMID:20655849

Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Starbird, C. A.; Maklashina, Elena; Sharma, Pankaj

The Escherichia coli Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the E. coli QFR FrdA subunit. Using a ΔsdhE E. coli strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdAE245more » residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdAE245Q have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdAE245 variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdAE245 residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.« less

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier ProteinTethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus

DOE PAGES

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan; ...

2016-08-25

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

Cloning and Characterization of Lxr and Srebp1, and Their Potential Roles in Regulation of LC-PUFA Biosynthesis in Rabbitfish Siganus canaliculatus.

PubMed

Zhang, Qinghao; You, Cuihong; Liu, Fang; Zhu, Wendi; Wang, Shuqi; Xie, Dizhi; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

2016-09-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability to biosynthesize C20-22 long-chain polyunsaturated fatty acid (LC-PUFA) from C18 PUFA precursors, which is generally absent or low in marine teleosts. Thus, understanding the molecular basis of LC-PUFA biosynthesis in rabbitfish will contribute to efforts aimed at optimizing LC-PUFA biosynthesis in teleosts, especially marine species. In the present study, the importance of the transcription factors liver X receptor (Lxr) and sterol regulatory element-binding protein 1 (Srebp1) in regulation of LC-PUFA biosynthesis in rabbitfish was investigated. First, full-length cDNA of Lxr and Srebp1 were cloned and characterized. The Lxr mRNA displayed a ubiquitous tissue expression pattern while Srebp1 was highly expressed in eyes, brain and intestine. In rabbitfish primary hepatocytes treated with Lxr agonist T0901317, the expression of Lxr and Srebp1 was activated, accompanied by elevated mRNA levels of Δ4 and Δ6/Δ5 fatty acyl desaturase (Fad), key enzymes of LC-PUFA biosynthesis, as well as peroxisome proliferator-activated receptor γ (PPARγ). In addition, Srebp1 displayed higher expression levels in liver of rabbitfish fed a vegetable oil diet or reared at 10 ppt salinity, which were conditions reported to increase the liver expression of Δ4 and Δ6/Δ5 Fad and LC-PUFA biosynthetic ability, than fish fed a fish oil diet or reared at 32 ppt, respectively. These results suggested that Lxr and Srebp1 are involved in regulation of LC-PUFA biosynthesis probably by promoting the expression of two Fad in rabbitfish liver, which, to our knowledge, is the first report in marine teleosts.

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca2+-ATPase: kinetic pathway and effects of methionine oxidation

PubMed Central

Slaughter, Brian D.; Bieber Urbauer, Ramona J.; Urbauer, Jeffrey L.; Johnson, Carey K.

2008-01-01

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma-membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide (C28W(1b)) corresponding to the CaM binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or on the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism where an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the lower effectiveness of oxidized CaM in activating the Ca2+ pump. PMID:17343368

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

PubMed

Kadamur, Ganesh; Ross, Elliott M

2016-05-20

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

PubMed Central

Kadamur, Ganesh

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction.

PubMed

Zhang, Lin; Trncik, Christian; Andrade, Susana L A; Einsle, Oliver

2017-02-01

The copper-containing enzyme nitrous oxide reductase (N 2 OR) catalyzes the transformation of nitrous oxide (N 2 O) to dinitrogen (N 2 ) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites Cu A and Cu Z , and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N 2 O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg 2+ that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent. Copyright © 2016 Elsevier B.V. All rights reserved.

FADS1 FADS2 Gene Cluster, PUFA Intake and Blood Lipids in Children: Results from the GINIplus and LISAplus Studies

PubMed Central

Standl, Marie; Lattka, Eva; Stach, Barbara; Koletzko, Sibylle; Bauer, Carl-Peter; von Berg, Andrea; Berdel, Dietrich; Krämer, Ursula; Schaaf, Beate; Röder, Stefan; Herbarth, Olf; Buyken, Anette; Drogies, Tim; Thiery, Joachim; Koletzko, Berthold; Heinrich, Joachim

2012-01-01

Background Elevated cholesterol levels in children can be a risk factor for cardiovascular diseases in later life. In adults, it has been shown that blood lipid levels are strongly influenced by polymorphisms in the fatty acid desaturase (FADS) gene cluster in addition to nutritional and other exogenous and endogenous determinants. Our aim was to investigate whether lipid levels are determined by the FADS genotype already in children and whether this association interacts with dietary intake of n-3 fatty acids. Methods The analysis was based on data of 2006 children from two German prospective birth cohort studies. Total cholesterol, HDL, LDL and triglycerides were measured at 10 years of age. Six single nucleotide polymorphisms (SNPs) of the FADS gene cluster were genotyped. Dietary n-3 fatty acid intake was assessed by food frequency questionnaire. Linear regression modeling was used to assess the association between lipid levels, n-3 fatty acid intake and FADS genotype. Results Individuals carrying the homozygous minor allele had lower levels of total cholesterol [means ratio (MR) ranging from 0.96 (p = 0.0093) to 0.98 (p = 0.2949), depending on SNPs] and LDL [MR between 0.94 (p = 0.0179) and 0.97 (p = 0.2963)] compared to homozygous major allele carriers. Carriers of the heterozygous allele showed lower HDL levels [β between −0.04 (p = 0.0074) to −0.01 (p = 0.3318)] and higher triglyceride levels [MR ranging from 1.06 (p = 0.0065) to 1.07 (p = 0.0028)] compared to homozygous major allele carriers. A higher n-3 PUFA intake was associated with higher concentrations of total cholesterol, LDL, HDL and lower triglyceride levels, but these associations did not interact with the FADS1 FADS2 genotype. Conclusion Total cholesterol, HDL, LDL and triglyceride concentrations may be influenced by the FADS1 FADS2 genotype already in 10 year old children. Genetically determined blood lipid levels during childhood might differentially predispose individuals to the development of cardiovascular diseases later in life. PMID:22629455

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

PubMed

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Menon; S Wang

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, themore » switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.« less

Use of 5-deazaFAD to study hydrogen transfer in the D-amino acid oxidase reaction.

PubMed

Hersh, L B; Jorns, M S

1975-11-25

The apoprotein of hog kidney D-amino acid oxidase was reconstituted with 5-deazaflavin adenine dinucleotide (5-deazaFAD) to yield a protein which contains 1.5 mol of 5-deazaFAD/mol of enzyme. The deazaFAD-containing enzyme forms complexes with benzoate, 2-amino benzoate, and 4-aminobenzoate which are both qualitatively and quantitatively similar to those observed with native enzyme. The complex with 2-aminobenzoate exhibits a new long wavelength absorption band characteristic of a flavin charge-transfer complex. The reconstituted enzyme exhibits no activity when assayed by D-alanine oxidation. However, the bound chromophore can be reduced by alanine, phenylalanine, proline, methionine, and valine, but not by glutamate or aspartate, indicating the deazaFAD enzyme retains the substrate specificity of the native enzyme. Reduction of the enzyme by D-alanine exhibits a 1.6-fold deuterium isotope effect. Reoxidation of the reduced enzyme occurred in the presence of pyruvate plus ammonia, but not with pyruvate alone or ammonia alone. beta-Phenylpyruvate and alpha-ketobutyrate, but not alpha-ketoglutarate could replace pyruvate. Reduced enzyme isolated following reaction with [alpha-3H]alanine was found to contain 0.5 mol of tritium/mol of deazaFADH2. After denaturation of the tritium-labeled enzyme, the radioactivity was identified as deazaFADH2. Reaction of the reduced tritium-labeled enzyme with pyruvate plus ammonia prior to denaturation yields [alpha-3H]alanine and unlabeled deazaFAD. These results suggest that reduction and reoxidation of enzyme-bound deazaFAD involves the stereo-specific transfer of alpha-hydrogen from substrate to deazaFAD.

Evolutionary divergence of chloroplast FAD synthetase proteins

PubMed Central

2010-01-01

Background Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity. PMID:20955574

FATTY ACID DESATURASE4 of Arabidopsis encodes a protein distinct from characterized fatty acid desaturases.

PubMed

Gao, Jinpeng; Ajjawi, Imad; Manoli, Arthur; Sawin, Andrew; Xu, Changcheng; Froehlich, John E; Last, Robert L; Benning, Christoph

2009-12-01

Polar membrane glycerolipids occur in a mixture of molecular species defined by a polar head group and characteristic acyl groups esterified to a glycerol backbone. A molecular species of phosphatidylglycerol specific to chloroplasts of plants carries a Delta(3-trans) hexadecenoic acid in the sn-2 position of its core glyceryl moiety. The fad4-1 mutant of Arabidopsis thaliana missing this particular phosphatidylglycerol molecular species lacks the necessary fatty acid desaturase, or a component thereof. The overwhelming majority of acyl groups associated with membrane lipids in plants contains double bonds with a cis configuration. However, FAD4 is unusual because it is involved in the formation of a trans double bond introduced close to the carboxyl group of palmitic acid, which is specifically esterified to the sn-2 glyceryl carbon of phosphatidylglycerol. As a first step towards the analysis of this unusual desaturase reaction, the FAD4 gene was identified by mapping of the FAD4 locus and coexpression analysis with known lipid genes. FAD4 encodes a predicted integral membrane protein that appears to be unrelated to classic membrane bound fatty acid desaturases based on overall sequence conservation. However, the FAD4 protein contains two histidine motifs resembling those of metalloproteins such as fatty acid desaturases. FAD4 is targeted to the plastid. Overexpression of the cDNA in transgenic Arabidopsis led to increased accumulation of the Delta(3-trans) hexadecanoyl group in phosphatidylglycerol relative to wild type. Taken together these results are consistent with the hypothesis that FAD4 is the founding member of a novel class of fatty acid desaturases.

Determination of fatty acid composition in seed oil of rapeseed (Brassica napus L.) by mutated alleles of the FAD3 desaturase genes.

PubMed

Bocianowski, Jan; Mikołajczyk, Katarzyna; Bartkowiak-Broda, Iwona

2012-02-01

One of the goals in oilseed rape programs is to develop genotypes producing oil with low linolenic acid content (C18:3, ≤3%). Low linolenic mutant lines of canola rapeseed were obtained via chemical mutagenesis at the Plant Breeding and Acclimatization Institute - NRI, in Poznan, Poland, and allele-specific SNP markers were designed for monitoring of two statistically important single nucleotide polymorphisms detected by SNaPshot analysis in two FAD3 desaturase genes, BnaA.FAD3 and BnaC.FAD3, respectively. Strong negative correlation between the presence of mutant alleles of the genes and linolenic acid content was revealed by analysis of variance. In this paper we present detailed characteristics of the markers by estimation of the additive and dominance effects of the FAD3 genes with respect to particular fatty acid content in seed oil, as well as by calculation of the phenotypic variation of seed oil fatty acid composition accounted by particular allele-specific marker. The obtained percentage of variation in fatty acid composition was considerable only for linolenic acid content and equaled 35.6% for BnaA.FAD3 and 39.3% for BnaC.FAD3, whereas the total percentage of variation in linolenic acid content was 53.2% when accounted for mutations in both genes simultaneously. Our results revealed high specificity of the markers for effective monitoring of the wild-type and mutated alleles of the Brassica napus FAD3 desaturase genes in the low linolenic mutant recombinants in breeding programs.

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

PubMed

Danylovych, H V

2016-01-01

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

PubMed Central

Siaud, Nicolas; Lam, Isabel; Christ, Nicole; Schlacher, Katharina; Xia, Bing; Jasin, Maria

2011-01-01

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations. PMID:22194698

An Icelandic Version of McMasters Family Assessment Device (FAD)

ERIC Educational Resources Information Center

Juliusdottir, Gudlaug M.; Olafsdottir, Hrefna

2015-01-01

Purpose: An analysis of the psychometric properties of an Icelandic version of McMasters Family Assessment Device (FAD) was conducted in this study. Method: Two groups, clinical and nonclinical, comprising of 529 parents answered the FAD. The study examined the internal reliability and discriminant validity of the instrument in addition to…

The EFQM Excellence Model[R]: Higher Education's Latest Management Fad?

ERIC Educational Resources Information Center

Temple, Paul

2005-01-01

Robert Birnbaum argues that higher education tends to adopt management fads -- newly conceived techniques enjoying brief popularity but which fail to live up to their promoters claims at the point when the corporate sector and government are discarding them. Although fads may have failed in these sectors because of various reasons, their failure…

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

PubMed

Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

2013-10-15

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

The X-43A Flush Airdata Sensing System Flight Test Results

NASA Technical Reports Server (NTRS)

Baumann, Ethan; Pahle, Joseph W.; Davis, Mark; White, John Terry

2008-01-01

The National Aeronautics and Space Administration (NASA) has flight-tested a flush airdata sensing (FADS) system on the Hyper-X Research Vehicle (X-43A) at hypersonic speeds during the course of two successful flights. For this series of tests, the FADS system was calibrated to operate between Mach 3 and Mach 8, and flight test data was collected between Mach 1 and Mach 10. The FADS system acquired pressure data from surface-mounted ports and generated a real-time angle-of-attack (alpha) estimate on board the X-43A. The collected data were primarily intended to evaluate the FADS system performance, and the estimated alpha was used by the flight control algorithms on the X-43A for only a portion of the first successful flight. This paper provides an overview of the FADS system and alpha estimation algorithms, presents the in-flight alpha estimation algorithm performance, and provides comparisons to wind tunnel results and theory. Results indicate that the FADS system adequately estimated the alpha of the vehicle during the hypersonic portions of the two flights.

Characterization of 3-ketosteroid 9{alpha}-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis.

PubMed

Capyk, Jenna K; D'Angelo, Igor; Strynadka, Natalie C; Eltis, Lindsay D

2009-04-10

KshAB (3-Ketosteroid 9alpha-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O(2). Nevertheless, the reactivity of KshAB with O(2) was low in the presence of 1,4-androstadiene-3,17-dione, with a k(cat)/K(m)(O(2)) of 2450 +/- 80 m(-1) s(-1). The crystallographic structure of KshA, determined to 2.3A(,) revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance.

Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis*S⃞

PubMed Central

Capyk, Jenna K.; D'Angelo, Igor; Strynadka, Natalie C.; Eltis, Lindsay D.

2009-01-01

KshAB (3-Ketosteroid 9α-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O2. Nevertheless, the reactivity of KshAB with O2 was low in the presence of 1,4-androstadiene-3,17-dione, with a kcat/KmO2 of 2450 ± 80 m–1 s–1. The crystallographic structure of KshA, determined to 2.3Å, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance. PMID:19234303

The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions.

PubMed

Suginta, Wipa; Sirimontree, Paknisa; Sritho, Natchanok; Ohnuma, Takayuki; Fukamizo, Tamo

2016-12-01

Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β) 8 TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD Vh ChiA . The recombinant ChBD Vh ChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD Vh ChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBD Vh ChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis[W

PubMed Central

Aggarwal, Pooja; Das Gupta, Mainak; Joseph, Agnel Praveen; Chatterjee, Nirmalya; Srinivasan, N.; Nath, Utpal

2010-01-01

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an ∼60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix (bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors. PMID:20363772

In vitro/in vivo comparison of drug release and polymer erosion from biodegradable P(FAD-SA) polyanhydrides--a noninvasive approach by the combined use of electron paramagnetic resonance spectroscopy and nuclear magnetic resonance imaging.

PubMed

Mäder, K; Crémmilleux, Y; Domb, A J; Dunn, J F; Swartz, H M

1997-06-01

The purpose of this study was to compare drug release and polymer erosion from biodegradable P(FAD-SA) polyanhydrides in vitro and in vivo in real time and with minimal disturbance of the investigated system. P(FAD-SA) 20:80 and P(FAD-SA) 50:50 polymer tablets were loaded with the spin probe 3-carboxy-2,2,5,5-tetramethyl-pyrrollidine-1-oxyl (PCA) and implanted subcutaneously in the neck of rats or placed in 0.1 M phosphate buffer. 1.1 GHz EPR spectroscopy experiments and 7T MRI studies (T1 and T2 weighted) were performed. A front of water penetration was visible by MRI in vitro in the case of P(FAD-SA) 20:80, but not for P(FAD-SA) 50:50. For both polymers, the thickness of the tablets decreased with time and a insoluble, easy deformable residue remained. Important processes such as edema, deformation of the implant, encapsulation and bioresorption were observable by MRI in vivo. P(FAD-SA) 50:50 was almost entirely absorbed by day 44, whereas an encapsulated residue was found for P(FAD-SA) 20:80 after 65 days. The EPR studies gave direct evidence of a water penetration induced changes of the microenvironment inside the tablet. EPR signals were still detectable in P(FAD-SA) 20:80 implants after 65 days, while the nitroxide was released in vitro within 16 days. Important parameters and processes such as edema, deformation of the tablet, microviscosity inside the tablet and encapsulation can be monitored in real time by the combined use of the noninvasive techniques MRI and EPR leading to better understanding of the differences between the in vitro and in vivo situation.

Structural basis of redox-dependent substrate binding of protein disulfide isomerase

PubMed Central

Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

2015-01-01

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

PubMed

Gunawardana, Dilantha

2016-01-01

Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminalmore » Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.« less

Structure of the ACF7 EF-Hand-GAR Module and Delineation of Microtubule Binding Determinants.

PubMed

Lane, Thomas R; Fuchs, Elaine; Slep, Kevin C

2017-07-05

Spectraplakins are large molecules that cross-link F-actin and microtubules (MTs). Mutations in spectraplakins yield defective cell polarization, aberrant focal adhesion dynamics, and dystonia. We present the 2.8 Å crystal structure of the hACF7 EF1-EF2-GAR MT-binding module and delineate the GAR residues critical for MT binding. The EF1-EF2 and GAR domains are autonomous domains connected by a flexible linker. The EF1-EF2 domain is an EFβ-scaffold with two bound Ca 2+ ions that straddle an N-terminal α helix. The GAR domain has a unique α/β sandwich fold that coordinates Zn 2+ . While the EF1-EF2 domain is not sufficient for MT binding, the GAR domain is and likely enhances EF1-EF2-MT engagement. Residues in a conserved basic patch, distal to the GAR domain's Zn 2+ -binding site, mediate MT binding. Copyright © 2017 Elsevier Ltd. All rights reserved.

Specific-pathogen-free chickens vaccinated with a live FAdV-4 vaccine are fully protected against a severe challenge even in the absence of neutralizing antibodies.

PubMed

Schonewille, Esther; Jaspers, Ron; Paul, Guntram; Hess, Michael

2010-06-01

By adapting a very virulent fowl adenovirus serotype 4 (FAdV-4) to a fibroblast cell line (QT35) instead of growing the virus in chicken embryo liver cells or chicken kidney cells, it was possible to attenuate the virus. Birds infected with the attenuated virus (FAdV-4/QT35) on the first day of life expressed no adverse clinical signs and no mortality. Intramuscular challenge with the virulent virus grown on chicken embryo liver cells (FAdV-4/CEL) at 21 days of life induced high mortality in previously nonvaccinated birds, whereas none of the birds vaccinated at 1 day old with FAdV-4/QT35 died due to this challenge. Applying enzyme-linked immunosorbent assay and virus neutralization assay, only a weak antibody response could be detected in some birds following vaccination, a response that increased directly after challenge. Nonvaccinated birds displayed a delayed development of antibodies after challenge as compared to previously vaccinated birds. Even birds that did not develop a measurable neutralizing antibody titer prior to challenge were protected from the adverse effects of the virulent FAdV-4/CEL, a phenomenon not described so far for FAdVs. Altogether, the present investigation underlines that neutralizing antibodies are not needed to protect chickens against a severe infection with a virulent fowl adenovirus.

Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9

USDA-ARS?s Scientific Manuscript database

The CRISPR/Cas9 system is known for its precise and efficient gene-editing of a targeted region in a variety of organisms including plants. We targeted FAD2 gene region to perform CRISPR/Cas9 gene-editing in peanut. The FAD2 gene encodes fatty acid desaturase which catalyzes the conversion of oleic ...

Body Weight, Body Image, and Perception of Fad Diets in Adolescent Girls.

ERIC Educational Resources Information Center

Storz, Nancy S.; Greene, Walter H.

1983-01-01

Examined relationships among adolescent girls' (N=203) satisfaction with body weight, body image, and perception/use of fad diets. Subjects wanting to lose weight were placed into two groups based on amount of weight-loss desired and compared in terms of body image scores, ratings of fad diets, and frequency of using the diets. (JN)

Identification of QTLs associated with oil content and mapping FAD2 genes and their relative contribution to oil quality in peanut (Arachis hypogaea L.).

PubMed

Pandey, Manish K; Wang, Ming Li; Qiao, Lixian; Feng, Suping; Khera, Pawan; Wang, Hui; Tonnis, Brandon; Barkley, Noelle A; Wang, Jianping; Holbrook, C Corley; Culbreath, Albert K; Varshney, Rajeev K; Guo, Baozhu

2014-12-10

Peanut is one of the major source for human consumption worldwide and its seed contain approximately 50% oil. Improvement of oil content and quality traits (high oleic and low linoleic acid) in peanut could be accelerated by exploiting linked markers through molecular breeding. The objective of this study was to identify QTLs associated with oil content, and estimate relative contribution of FAD2 genes (ahFAD2A and ahFAD2B) to oil quality traits in two recombinant inbred line (RIL) populations. Improved genetic linkage maps were developed for S-population (SunOleic 97R × NC94022) with 206 (1780.6 cM) and T-population (Tifrunner × GT-C20) with 378 (2487.4 cM) marker loci. A total of 6 and 9 QTLs controlling oil content were identified in the S- and T-population, respectively. The contribution of each QTL towards oil content variation ranged from 3.07 to 10.23% in the S-population and from 3.93 to 14.07% in the T-population. The mapping positions for ahFAD2A (A sub-genome) and ahFAD2B (B sub-genome) genes were assigned on a09 and b09 linkage groups. The ahFAD2B gene (26.54%, 25.59% and 41.02% PVE) had higher phenotypic effect on oleic acid (C18:1), linoleic acid (C18:2), and oleic/linoleic acid ratio (O/L ratio) than ahFAD2A gene (8.08%, 6.86% and 3.78% PVE). The FAD2 genes had no effect on oil content. This study identified a total of 78 main-effect QTLs (M-QTLs) with up to 42.33% phenotypic variation (PVE) and 10 epistatic QTLs (E-QTLs) up to 3.31% PVE for oil content and quality traits. A total of 78 main-effect QTLs (M-QTLs) and 10 E-QTLs have been detected for oil content and oil quality traits. One major QTL (more than 10% PVE) was identified in both the populations for oil content with source alleles from NC94022 and GT-C20 parental genotypes. FAD2 genes showed high effect for oleic acid (C18:1), linoleic acid (C18:2), and O/L ratio while no effect on total oil content. The information on phenotypic effect of FAD2 genes for oleic acid, linoleic acid and O/L ratio, and oil content will be applied in breeding selection.

Biophysical basis of the binding of WWOX tumor suppressor to WBP1 and WBP2 adaptors.

PubMed

McDonald, Caleb B; Buffa, Laura; Bar-Mag, Tomer; Salah, Zaidoun; Bhat, Vikas; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Malhotra, Arun; Sudol, Marius; Aqeilan, Rami I; Nawaz, Zafar; Farooq, Amjad

2012-09-07

The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

PubMed

Devkota, Sujan; Joseph, Raji E; Boyken, Scott E; Fulton, D Bruce; Andreotti, Amy H

2017-06-13

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP 3 . By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

[Desaturases of fatty acids (FADS) and their physiological and clinical implication].

PubMed

Žák, Aleš; Slabý, Adolf; Tvrzická, Eva; Jáchymová, Marie; Macášek, Jaroslav; Vecka, Marek; Zeman, Miroslav; Staňková, Barbora

States associated with insulin resistance, as overweight/obesity, type 2 diabetes mellitus (DM2), cardiovascular diseases (CVD), some cancers and neuropsychiatric diseases are characterized with a decrease of long-chain polyunsaturated fatty acids (LC-PUFA) levels. Amounts of LC-PUFA depend on the exogenous intake of their precursors [linoleic (LA) and α-linolenic acid (ALA)] and by rate of their metabolism, which is influenced by activities of enzymes, such as Δ6-desaturase (D6D, FADS2), D5D, FADS1, elongases (Elovl2, -5, 6).Altered activities of D5D/D6D were described in plenty of diseases, e.g. neuropsychiatric (depressive disorders, bipolar disorder, dementia), metabolic (obesity, metabolic syndrome, DM2) and cardiovascular diseases (arterial hypertension, coronary heart disease), inflammatory states and allergy (Crohns disease, atopic eczema) or some malignancies. Similar results were obtained in studies dealing with the associations between genotypes/haplotypes of FADS1/FADS2 and above mentioned diseases, or interactions of dietary intake of LA and ALA on one hand and of the polymorphisms of minor allels of FADS1/FADS2, usually characterized by lower activities, on the other hand.The decrease of the desaturases activities leads to decreased concentrations of products with concomitant increased concentrations of substrates. Associations of some SNP FADS with coronary heart disease, concentrations of plasma lipids, oxidative stress, glucose homeostasis, and inflammatory reaction, were described. Experimental studies on animal models and occurrence of rare diseases, associated with missing or with marked fall activities of D5D/D6D emphasized the significance of desaturases for healthy development of organism as well as for pathogenesis of some disease.

Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids*

PubMed Central

Okada, Shoko; Zhou, Xue-Rong; Damcevski, Katherine; Gibb, Nerida; Wood, Craig; Hamberg, Mats; Haritos, Victoria S.

2013-01-01

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family. PMID:24062307

Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

PubMed

Okada, Shoko; Zhou, Xue-Rong; Damcevski, Katherine; Gibb, Nerida; Wood, Craig; Hamberg, Mats; Haritos, Victoria S

2013-11-08

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.

High intensity focused ultrasound in the treatment of breast fibroadenomata: results of the HIFU-F trial.

PubMed

Peek, M C L; Ahmed, M; Scudder, J; Baker, R; Pinder, S E; Douek, M

2016-12-01

Breast fibroadenomata (FAD) are the most common breast lumps in women. High intensity focused ultrasound (HIFU) is a non-invasive ablative technique that can be used to treat FAD but is associated with prolonged treatment times. In the HIFU-F trial, we evaluated the change in volume over time with circumferential HIFU treatment of FAD and compared this to no treatment. Patients ≥18 years, diagnosed with symptomatic, palpable FAD, visible on ultrasound (US) were recruited. Twenty patients were treated using US-guided HIFU under local anaesthesia. Another 20 participants underwent an US 6 months after diagnosis. Outcome measures included: reduction in treatment time compared to whole lesion ablation; feasibility to achieve a 50% reduction in volume after 6 months; decrease in volume compared to a control group and reduction in symptoms. Circumferential ablation reduced the mean treatment time by 37.5% (SD 20.1%) compared to whole lesion ablation. US demonstrated a significant mean reduction in FAD volume of 43.5% (SD 38.8%; p = 0.016, paired t-test) in the HIFU group compared to 4.6% (SD 46.0%; p = 0.530) in the control group after 6 months. This mean reduction in FAD volume between the two groups was significant in favour of the HIFU group (p = 0.002, grouped t-test). Pre-treatment pain completely resolved in 6 out of 8 patients 6 months post-treatment. Circumferential HIFU ablation of FAD is feasible, with a significant reduction in pain and volume compared to control participants. It provides a simple, non-invasive, outpatient-based alternative to surgical excision for FAD.

Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the Developing Embryo of Pecan (Carya illinoinensis).

PubMed

Huang, Ruimin; Huang, Youjun; Sun, Zhichao; Huang, Jianqin; Wang, Zhengjia

2017-05-24

Pecan (Carya illinoinensis) is an important woody tree species because of the high content of healthy oil in its nut. Thus far, the pathways and key genes related to oil biosynthesis in developing pecan seeds remain largely unclear. Our analyses revealed that mature pecan embryo accumulated more than 80% oil, in which 90% was unsaturated fatty acids with abundant oleic acid. RNA sequencing generated 84,643 unigenes in three cDNA libraries prepared from pecan embryos collected at 105, 120, and 165 days after flowering (DAF). We identified 153 unigenes associated with lipid biosynthesis, including 107 unigenes for fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, 7 for oil bodies, and 5 for transcription factors involved in oil synthesis. The genes associated with fatty acid synthesis were the most abundantly expressed genes at 120 DAF. Additionally, the biosynthesis of oil began to increase while crude fat contents increased from 16.61 to 74.45% (165 DAF). We identified four SAD, two FAD2, one FAD6, two FAD7, and two FAD8 unigenes responsible for unsaturated fatty acid biosynthesis. However, FAD3 homologues were not detected. Consequently, we inferred that the linolenic acid in developing pecan embryos is generated by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic reticula. During pecan embryo development, different unigenes are expressed for plastidial and cytosolic glycolysis. Plastidial glycolysis is more relevant to lipid synthesis than cytosolic glycolysis. The 18 most important genes associated with lipid biosynthesis were evaluated in five stages of developing embryos using quantitative PCR (qPCR). The qPCR data were well consistent with their expression in transcriptomic analyses. Our data would be important for the metabolic engineering of pecans to increase oil contents and modify fatty acid composition.

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed.

PubMed

Liu, Hua Liang; Yin, Zhi Jie; Xiao, Li; Xu, Yi Nong; Qu, Le Qing

2012-05-01

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g⁻¹ to 8.57 mg g⁻¹ and 10.06 mg g⁻¹, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.

Cognitive function in adolescence: testing for interactions between breast-feeding and FADS2 polymorphisms.

PubMed

Martin, Nicolas W; Benyamin, Beben; Hansell, Narelle K; Montgomery, Grant W; Martin, Nicholas G; Wright, Margaret J; Bates, Timothy C

2011-01-01

Breast-fed C-allele carriers of the rs174575 single nucleotide polymorphism in the fatty acyl desaturase 2 (FADS2) gene have been reported to show a 6.4 to 7 IQ point advantage over formula-fed C-allele carriers, with no effect of breast-feeding in GG carriers. An Australian sample was examined to determine if an interaction between breast-feeding and the rs174575 single nucleotide polymorphism had any effect on IQ. This hypothesis was tested in more than 700 families of adolescent twins assessed for IQ and breast-feeding, birth weight, and FADS2 polymorphisms, and parental socioeconomic status and education, and maternal FADS2 status. No significant evidence for a moderating effect on IQ of rs174575 C-carrier status and breast-feeding was found, and there no effects of maternal FADS2 status on offspring IQ. In addition, no main effects of any FADS2 polymorphisms on IQ were found when the genotype was kept as two-homozygote and one-heterozygote categories and indeed no evidence for effects of breast-feeding on IQ scores after controlling for parental socioeconomic status and education. The investigation was extended to two additional FADS2 polymorphisms (rs1535 and rs174583), but again, although these polymorphisms code alleles affecting fatty acid metabolism, no main or interaction effects were found on IQ. These results support the view that apparent effects of breast-feeding on IQ reflect differential likelihood of breast-feeding as a function of parental education and did not support the predicted interaction effect of FADS2 and breast-feeding on IQ. Copyright © 2011 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.

Omega-3 fatty acid deficiency selectively up-regulates delta6-desaturase expression and activity indices in rat liver: prevention by normalization of omega-3 fatty acid status.

PubMed

Hofacer, Rylon; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Magrisso, I Jack; Benoit, Stephen C; McNamara, Robert K

2011-09-01

This study investigated the effects of perinatal dietary omega-3 (n-3) fatty acid depletion and subsequent repletion on the expression of genes that regulate long-chain (LC) polyunsaturated fatty acid biosynthesis in rat liver and brain. It was hypothesized that chronic n-3 fatty acid deficiency would increase liver Fads1 and Fads2 messenger RNA (mRNA) expression/activity and that n-3 fatty acid repletion would normalize this response. Adult rats fed the n-3-free diet during perinatal development exhibited significantly lower erythrocyte, liver, and frontal cortex LCn-3 fatty acid composition and reciprocal elevations in LC omega-6 (n-6) fatty acid composition compared with controls (CONs) and repleted rats. Liver Fads2, but not Fads1, Elovl2, or Elovl5, mRNA expression was significantly greater in n-3-deficient (DEF) rats compared with CONs and was partially normalized in repleted rats. The liver 18:3n-6/18:2n-6 ratio, an index of delta6-desturase activity, was significantly greater in DEF rats compared with CON and repleted rats and was positively correlated with Fads2 mRNA expression among all rats. The liver 18:3n-6/18:2n-6 ratio, but not Fads2 mRNA expression, was also positively correlated with erythrocyte and frontal cortex LCn-6 fatty acid compositions. Neither Fads1 or Fads2 mRNA expression was altered in brain cortex of DEF rats. These results confirm previous findings that liver, but not brain, delta6-desaturase expression and activity indices are negatively regulated by dietary n-3 fatty acids. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seong K., E-mail: skim1@lsuhsc.edu; Kim, Seongman; Dai Gan

2011-09-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% ofmore » control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: > We examine the functional domains of IR2P that mediates negative regulation. > IR2P inhibits at the transcriptional level. > DNA-binding mutant or TFIIB-binding mutant fails to inhibit. > C-terminal aa 707 to 1116 are required for full inhibition. > Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.« less

Solution structure of the Big domain from Streptococcus pneumoniae reveals a novel Ca2+-binding module

PubMed Central

Wang, Tao; Zhang, Jiahai; Zhang, Xuecheng; Xu, Chao; Tu, Xiaoming

2013-01-01

Streptococcus pneumoniae is a pathogen causing acute respiratory infection, otitis media and some other severe diseases in human. In this study, the solution structure of a bacterial immunoglobulin-like (Big) domain from a putative S. pneumoniae surface protein SP0498 was determined by NMR spectroscopy. SP0498 Big domain adopts an eight-β-strand barrel-like fold, which is different in some aspects from the two-sheet sandwich-like fold of the canonical Ig-like domains. Intriguingly, we identified that the SP0498 Big domain was a Ca2+ binding domain. The structure of the Big domain is different from those of the well known Ca2+ binding domains, therefore revealing a novel Ca2+-binding module. Furthermore, we identified the critical residues responsible for the binding to Ca2+. We are the first to report the interactions between the Big domain and Ca2+ in terms of structure, suggesting an important role of the Big domain in many essential calcium-dependent cellular processes such as pathogenesis. PMID:23326635

Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose.

PubMed

Tomoo, Koji; Miki, Yasuhiro; Morioka, Hideaki; Seike, Kiho; Ishida, Toshimasa; Ikenishi, Sadao; Miyamoto, Katsushiro; Hasegawa, Tomokazu; Yamano, Akihito; Hamada, Kensaku; Tsujibo, Hiroshi

2017-06-01

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Exploring the molecular basis of RNA recognition by the dimeric RNA-binding protein via molecular simulation methods.

PubMed

Chang, Shan; Zhang, Da-Wei; Xu, Lei; Wan, Hua; Hou, Ting-Jun; Kong, Ren

2016-11-01

RNA-binding protein with multiple splicing (RBPMS) is critical for axon guidance, smooth muscle plasticity, and regulation of cancer cell proliferation and migration. Recently, different states of the RNA-recognition motif (RRM) of RBPMS, one in its free form and another in complex with CAC-containing RNA, were determined by X-ray crystallography. In this article, the free RRM domain, its wild type complex and 2 mutant complex systems are studied by molecular dynamics (MD) simulations. Through comparison of free RRM domain and complex systems, it's found that the RNA binding facilitates stabilizing the RNA-binding interface of RRM domain, especially the C-terminal loop. Although both R38Q and T103A/K104A mutations reduce the binding affinity of RRM domain and RNA, the underlining mechanisms are different. Principal component analysis (PCA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore the dynamical and recognition mechanisms of RRM domain and RNA. R38Q mutation is positioned on the homodimerization interface and mainly induces the large fluctuations of RRM domains. This mutation does not directly act on the RNA-binding interface, but some interfacial hydrogen bonds are weakened. In contrast, T103A/K104A mutations are located on the RNA-binding interface of RRM domain. These mutations obviously break most of high occupancy hydrogen bonds in the RNA-binding interface. Meanwhile, the key interfacial residues lose their favorable energy contributions upon RNA binding. The ranking of calculated binding energies in 3 complex systems is well consistent with that of experimental binding affinities. These results will be helpful in understanding the RNA recognition mechanisms of RRM domain.

Exploring the molecular basis of RNA recognition by the dimeric RNA-binding protein via molecular simulation methods

PubMed Central

Chang, Shan; Zhang, Da-Wei; Xu, Lei; Wan, Hua; Hou, Ting-Jun; Kong, Ren

2016-01-01

ABSTRACT RNA-binding protein with multiple splicing (RBPMS) is critical for axon guidance, smooth muscle plasticity, and regulation of cancer cell proliferation and migration. Recently, different states of the RNA-recognition motif (RRM) of RBPMS, one in its free form and another in complex with CAC-containing RNA, were determined by X-ray crystallography. In this article, the free RRM domain, its wild type complex and 2 mutant complex systems are studied by molecular dynamics (MD) simulations. Through comparison of free RRM domain and complex systems, it's found that the RNA binding facilitates stabilizing the RNA-binding interface of RRM domain, especially the C-terminal loop. Although both R38Q and T103A/K104A mutations reduce the binding affinity of RRM domain and RNA, the underlining mechanisms are different. Principal component analysis (PCA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore the dynamical and recognition mechanisms of RRM domain and RNA. R38Q mutation is positioned on the homodimerization interface and mainly induces the large fluctuations of RRM domains. This mutation does not directly act on the RNA-binding interface, but some interfacial hydrogen bonds are weakened. In contrast, T103A/K104A mutations are located on the RNA-binding interface of RRM domain. These mutations obviously break most of high occupancy hydrogen bonds in the RNA-binding interface. Meanwhile, the key interfacial residues lose their favorable energy contributions upon RNA binding. The ranking of calculated binding energies in 3 complex systems is well consistent with that of experimental binding affinities. These results will be helpful in understanding the RNA recognition mechanisms of RRM domain. PMID:27592836

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this studymore » we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.« less

Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.

PubMed

Murciano-Calles, Javier; McLaughlin, Megan E; Erijman, Ariel; Hooda, Yogesh; Chakravorty, Nishant; Martinez, Jose C; Shifman, Julia M; Sidhu, Sachdev S

2014-10-23

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

PubMed

Xu, Emma-Ruoqi; Blythe, Emily E; Fischer, Gerhard; Hyvönen, Marko

2017-07-28

Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

NASA Astrophysics Data System (ADS)

Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

2014-01-01

The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

2000-01-01

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

PubMed Central

Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

2017-01-01

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains. PMID:24107129

Structural and functional analysis of the YAP-binding domain of human TEAD2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.

2010-06-15

The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like {beta}-sandwich fold with two extra helix-turn-helixmore » inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.« less

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

NASA Technical Reports Server (NTRS)

Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

2000-01-01

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

PubMed

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

[Effect of flavin adenine dinucleotide on ultraviolet B induced damage in cultured human corneal epithelial cells].

PubMed

Sakamoto, Asuka; Nakamura, Masatsugu

2012-01-01

This study evaluated the effects of flavin adenine dinucleotide (FAD) on ultraviolet B (UV-B)-induced damage in cultured human corneal epithelial (HCE-T) cells. The cultured HCE-T cells were treated with 0.003125-0.05% FAD before exposure to 80 mJ/cm2 UV-B. Cell viability was measured 24 h after UV-B irradiation using the MTS assay. Reactive oxygen species (ROS) were detected 30 min after UV-B irradiation using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Apoptosis was evaluated 4 h after UV-B irradiation in the caspase-3/7 activity assay. UV-B irradiation reduced cell viability and stimulated ROS production and caspase-3/7 activity in HCE-T cells. Pretreatment of UV-B irradiated HCE-T cells with FAD significantly attenuated cell viability reduction and inhibited the stimulation of both ROS production and caspase-3/7 activity due to UV-B exposure compared with those with vehicle (0% FAD). These results clarified that FAD inhibits ROS-mediated apoptosis by UV-B irradiation in HCE-T cells and suggest that FAD may be effective as a radical scavenger in UV-B-induced corneal damage.

Evidence that norflurazon affects chloroplast lipid unsaturation in soybean leaves (Glycine max L.).

PubMed

Abrous-Belbachir, Ouzna; De Paepe, Rosine; Trémolières, Antoine; Mathieu, Chantal; Ad, Fatiha; Benhassaine-Kesri, Ghouziel

2009-12-09

Norflurazon is a bleaching herbicide known to block carotenoid biosynthesis by inhibiting phytoene desaturase activity. Soybean plants were treated with norflurazon, and we examined the effects on the desaturation of lipid molecular species in leaves using ammonium [1-(14)C] oleate labeling. In monogalactosyldiacylglycerol (MGDG), the main chloroplast lipid, a decrease in 18:3/18:3 molecular species and an increase in its precursors 18:2/18:3 and 18:2/18:2 were observed suggesting that the omega(3) FAD7 desaturase activity in planta was inhibited by norflurazon. The in vitro activity of MGDG synthase was also inhibited by 69%. In contrast, the amount of 18:3/18:3 molecular species of phosphatidylcholine (PC) in the extraplastid compartment increased. The observed increase in in vitro lysoPC-acyltransferase activity and activation of desaturation of [1-(14)C] oleate suggest that extraplastid omega(3)FAD3 desaturase was activated. Analysis of the expression of omega(3) FAD3 and omega(3) FAD7 genes in norflurazon treated plants indicate that omega(3) FAD7 and omega(3) FAD3 desaturases are controlled at the post-transcriptional level.

The effect of fermented apple diet supplementation on the growth performance and meat quality in finishing pigs.

PubMed

Lee, Sung Dae; Kim, Hoi Yun; Jung, Hyun Jung; Ji, Sang Yun; Chowdappa, Rekha; Ha, Ji Hee; Song, Young Min; Park, Jun Cheol; Moon, Hong Kil; Kim, In Cheul

2009-02-01

The objective of the present study was to investigate the effect of fermented apple diet (FAD) supplementation on the growth performance and meat quality in finishing Berkshires. The FAD was made from dropped apple mixed with rice bran and barley bran. Until 81 +/- 1 kg live weight at 133 +/- 1 days, the animals were fed a growing diet, after which experimental samples were fixed at 0, 2, 4 and 6% FAD as C, T1, T2 and T3 in the finishing diets. Growth performance, ADG, ADFI and feed efficiency were improved in T1 than other groups. In carcass parameters, carcass weight was higher (P < 0.05) in T1 than in other groups. In meat quality, moisture and crude protein contents decreased (P < 0.05) by addition of FAD. pH(24) and WHC were higher (P < 0.05) in T1 than other groups. In sensory evaluation, marbling of fresh meat and tenderness, juiciness, flavor and overall acceptability of cooked meat were improved by the addition of FAD. According to the results of our experiment, FAD can be used for improvement of meat quality parameters.

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait

PubMed Central

2010-01-01

Background The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. Results Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. Conclusions We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation of high oleic acid soybeans provide a framework to efficiently develop soybean varieties to meet changing market demands. PMID:20828382

Exploration of the perceived and actual benefits of omega-3 fatty acids and the impact of FADS1 and FADS2 genetic information on dietary intake and blood levels of EPA and DHA.

PubMed

Roke, Kaitlin

2017-03-01

From a global health perspective, increased intake of omega-3 fatty acids (FAs), in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are beneficial for human health. However, the consumption of EPA- and DHA-rich foods such as fatty fish is low in the Western diet. Therefore, finding new ways to motivate people to increase their consumption of omega-3 FAs is essential. To find effective ways to motivate individuals, understanding people's awareness of omega-3 FAs and how they obtain their knowledge about nutrition and health is critical. Consequently, we developed an online survey to assess awareness and self-reported intake of omega-3 FAs and supplements in young adults. EPA and DHA are also produced endogenously to a limited extent through a pathway regulated by fatty acid desaturase 1 and 2 (FADS1 and FADS2) genes. Of relevance, single nucleotide polymorphisms (SNPs) in the FADS genes influence levels of omega-3 FAs, where minor allele carriers have lower levels compared with major allele carriers. Accordingly, we conducted a clinical trial to investigate FA levels in response to dietary EPA and DHA supplementation in young adults stratified by SNPs in FADS1 and FADS2. The level of reported awareness of omega-3 terminology varied depending on an individual's field of study and thus providing all participants with the same set of nutrition information could be an effective tool to increase knowledge and motivate behaviour change. Additionally, the variation in FA levels in accordance to SNPs in FADS1 and FADS2 could be used to create tailored nutritional recommendations which may improve lifestyle habits. The results discovered in the first 2 studies regarding awareness of omega-3 FAs and genetic variation were subsequently used to design a nutrigenetics intervention in young adults. Individuals who received their FADS1 genetic information were more aware of different omega-3 FAs and reported fewer barriers to their consumption by the end of the study, compared with those who did not receive their personal genetic information. All participants increased their intake of EPA and DHA, which was reflected in the analyses of red blood cells. Overall, this thesis demonstrates the power of combining nutritional and genetic information as motivators to increase omega-3 consumption.

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait.

PubMed

Pham, Anh-Tung; Lee, Jeong-Dong; Shannon, J Grover; Bilyeu, Kristin D

2010-09-09

The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation of high oleic acid soybeans provide a framework to efficiently develop soybean varieties to meet changing market demands.

The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces

PubMed Central

Wong, Edmond; Vaaje-Kolstad, Gustav; Ghosh, Avishek; Hurtado-Guerrero, Ramon; Konarev, Peter V.; Ibrahim, Adel F. M.; Svergun, Dmitri I.; Eijsink, Vincent G. H.; Chatterjee, Nabendu S.; van Aalten, Daan M. F.

2012-01-01

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons. PMID:22253590

Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle

2015-10-15

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM hasmore » several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 {angstrom} to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.« less

Comparative Genomics of Regulation of Fatty Acid and Branched-chain Amino Acid Utilization in Proteobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kazakov, Alexey E.; Rodionov, Dmitry A.; Arkin, Adam Paul

2008-10-31

Bacteria can use branched-chain amino acids (ILV, i.e. isoleucine, leucine, valine) and fatty acids (FA) as sole carbon and energy sources convering ILV into acetyl-CoA, propanoyl-CoA and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR and GntR families binding tomore » eleven distinct DNA motifs. The ILV degradation genes in gamma- and beta-proteobacteria are mainly regulated by anovel regulator from the MerR family (e.g., LiuR in Pseudomonas aeruginosa) (40 species), in addition, the TetR-type regulator LiuQ was identified in some beta-proteobacteria (8 species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in the Shewanella species. The FA degradation genes are controlled by four regulators including FadR in gamma-proteobacteria (34 species), PsrA in gamma- and beta-proteobacteria (45 species), FadP in beta-proteobacteria (14 species), and LiuR orthologs in alpha-proteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from the functional and evolutionary points of view.« less

N-terminus determines activity and specificity of styrene monooxygenase reductases.

PubMed

Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

2017-12-01

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinant spider silk genetically functionalized with affinity domains.

PubMed

Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

2014-05-12

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

NASA Technical Reports Server (NTRS)

Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

1995-01-01

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

Genetic and epigenetic transgenerational implications related to omega-3 fatty acids. Part II: maternal FADS2 rs174575 genotype and DNA methylation predict toddler cognitive performance.

PubMed

Cheatham, Carol L; Lupu, Daniel S; Niculescu, Mihai D

2015-11-01

Maternal transfer of fatty acids is important to fetal brain development. The prenatal environment may differentially affect the substrates supporting declarative memory abilities, as the level of fatty acids transferred across the placenta may be affected by the maternal fatty acid desaturase 2 (FADS2) rs174575 single nucleotide polymorphism. In this study, we hypothesized that toddler and maternal rs174575 genotype and FADS2 promoter methylation would be related to the toddlers' declarative memory performance. Seventy-one 16-month-old toddlers participated in an imitation paradigm designed to test immediate and long-term declarative memory abilities. FADS2 rs174575 genotype was determined and FADS2 promoter methylation was quantified from blood by bisulfite pyrosequencing for the toddlers and their natural mothers. Toddlers of GG mothers at the FADS2 rs174575 single nucleotide polymorphism did not perform as well on memory assessments as toddlers of CC or CG mothers when controlling for plasma α-linolenic acid and child genotype. Toddler methylation status was related to immediate memory performance, whereas maternal methylation status was related to delayed memory performance. Thus, prenatal experience and maternal FADS2 status have a pervasive, long-lasting influence on the brain development of the offspring, but as the postnatal environment becomes more primary, the offsprings' own biology begins to have an effect. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydrogen peroxide produced by glucose oxidase affects the performance of laccase cathodes in glucose/oxygen fuel cells: FAD-dependent glucose dehydrogenase as a replacement.

PubMed

Milton, Ross D; Giroud, Fabien; Thumser, Alfred E; Minteer, Shelley D; Slade, Robert C T

2013-11-28

Hydrogen peroxide production by glucose oxidase (GOx) and its negative effect on laccase performance have been studied. Simultaneously, FAD-dependent glucose dehydrogenase (FAD-GDH), an O2-insensitive enzyme, has been evaluated as a substitute. Experiments focused on determining the effect of the side reaction of GOx between its natural electron acceptor O2 (consumed) and hydrogen peroxide (produced) in the electrolyte. Firstly, oxygen consumption was investigated by both GOx and FAD-GDH in the presence of substrate. Relatively high electrocatalytic currents were obtained with both enzymes. O2 consumption was observed with immobilized GOx only, whilst O2 concentration remained stable for the FAD-GDH. Dissolved oxygen depletion effects on laccase electrode performances were investigated with both an oxidizing and a reducing electrode immersed in a single compartment. In the presence of glucose, dramatic decreases in cathodic currents were recorded when laccase electrodes were combined with a GOx-based electrode only. Furthermore, it appeared that the major loss of performance of the cathode was due to the increase of H2O2 concentration in the bulk solution induced laccase inhibition. 24 h stability experiments suggest that the use of O2-insensitive FAD-GDH as to obviate in situ peroxide production by GOx is effective. Open-circuit potentials of 0.66 ± 0.03 V and power densities of 122.2 ± 5.8 μW cm(-2) were observed for FAD-GDH/laccase biofuel cells.

A small-scale open-label study of the treatment of canine flea allergy dermatitis with fluralaner.

PubMed

Fisara, Petr; Shipstone, Michael; von Berky, Andrew; von Berky, Janet

2015-12-01

Fluralaner is an isoxazoline systemic insecticide and acaricide that provides persistent flea-killing activity on dogs for 12 weeks. European and US field studies have shown that fluralaner treatment alleviates the signs of flea allergy dermatitis (FAD) in client-owned dogs. To assess the clinical response in FAD affected dogs over the 12-week period following a single oral fluralaner treatment. Twenty client-owned dogs were diagnosed with FAD on the basis of compatible clinical signs and a positive response in flea antigen tests, using intradermal and or serological methods. An open-label small-scale study with all dogs receiving a single oral fluralaner treatment. All enrolled dogs were diagnosed with FAD and then clinically monitored at 4-week intervals for 12 weeks. Twenty dogs completed the study. All dogs were flea-free at all post-treatment assessments except for one dog that had a single flea at the first post-enrollment assessment at 4 weeks. At the 4-week post-treatment assessment active FAD signs had resolved in all dogs; at 8 weeks post-treatment, two dogs showed mild signs. All clinical signs of FAD had resolved at the final assessment of 12 weeks after treatment. A single administration of fluralaner alleviated or resolved signs associated with FAD in all treated dogs over the recommended 12-week treatment period. © 2015 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of Intervet Australia Pty Ltd.

Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

PubMed

Haun, William; Coffman, Andrew; Clasen, Benjamin M; Demorest, Zachary L; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre; Cedrone, Frederic; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

2014-09-01

Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

PubMed

Lerner, D R; Raikhel, N V

1992-06-05

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.

Intramolecular interactions regulate SAP97 binding to GKAP

PubMed Central

Wu, Hongju; Reissner, Carsten; Kuhlendahl, Sven; Coblentz, Blake; Reuver, Susanne; Kindler, Stefan; Gundelfinger, Eckart D.; Garner, Craig C.

2000-01-01

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP–GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners. PMID:11060025

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

PubMed

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

Bio-nanocapsules displaying various immunoglobulins as an active targeting-based drug delivery system.

PubMed

Tatematsu, Kenji; Iijima, Masumi; Yoshimoto, Nobuo; Nakai, Tadashi; Okajima, Toshihide; Kuroda, Shun'ichi

2016-04-15

The bio-nanocapsule (BNC) is an approximately 30-nm particle comprising the hepatitis B virus (HBV) envelope L protein and a lipid bilayer. The L protein harbors the HBV-derived infection machinery; therefore, BNC can encapsulate payloads such as drugs, nucleic acids, and proteins and deliver them into human hepatocytes specifically in vitro and in vivo. To diversify the possible functions of BNC, we generated ZZ-BNC by replacing the domain indispensable for the human hepatotrophic property of BNC (N-terminal region of L protein) with the tandem form of the IgG Fc-binding Z domain of Staphylococcus aureus protein A. Thus, the ZZ-BNC is an active targeting-based drug delivery system (DDS) nanocarrier that depends on the specificity of the IgGs displayed. However, the Z domain limits the animal species and subtypes of IgGs that can be displayed on ZZ-BNC. In this study, we introduced into BNC an Ig κ light chain-binding B1 domain of Finegoldia magna protein L (protein-L B1 domain) and an Ig Fc-binding C2 domain of Streptococcus species protein G (protein-G C2 domain) to produce LG-BNC. The LL-BNC was constructed in a similar way using a tandem form of the protein-L B1 domain. Both LG-BNC and LL-BNC could display rat IgGs, mouse IgG1, human IgG3, and human IgM, all of which not binding to ZZ-BNC, and accumulate in target cells in an antibody specificity-dependent manner. Thus, these BNCs could display a broad spectrum of Igs, significantly improving the prospects for BNCs as active targeting-based DDS nanocarriers. We previously reported that ZZ-BNC, bio-nanocapsule deploying the IgG-binding Z domain of protein A, could display cell-specific antibody in an oriented immobilization manner, and act as an active targeting-based DDS nanocarrier. Since the Z domain can only bind to limited types of Igs, we generated BNCs deploying other Ig-binding domains: LL-BNC harboring the tandem form of Ig-binding domain of protein L, and LG-BNC harboring the Ig binding domains of protein L and protein G sequentially. Both BNCs could display a broader spectrum of Igs than does the ZZ-BNC. When these BNCs displayed anti-CD11c IgG or anti-EGFR IgG, both of which cannot bind to Z domain, they could bind to and then enter their respective target cells. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides*

PubMed Central

Mitra, Sharmistha; Traughber, C. Alicia; Brannon, Mary K.; Gomez, Stephanie; Capelluto, Daniel G. S.

2013-01-01

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways. PMID:23880770

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

PubMed

Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

2010-11-05

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« less

Identification of a Hydrophobic Cleft in the LytTR Domain of AgrA as a Locus for Small Molecule Interactions that Inhibit DNA Binding

PubMed Central

Leonard, Paul G.; Bezar, Ian F.; Sidote, David J.; Stock, Ann M.

2012-01-01

The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA-binding activity. Using NMR, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA-binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds. PMID:23181972

Biophysical Basis of the Binding of WWOX Tumor Suppressor to WBP1 and WBP2 Adaptors

PubMed Central

McDonald, Caleb B.; Buffa, Laura; Bar-Mag, Tomer; Salah, Zaidoun; Bhat, Vikas; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Malhotra, Arun; Sudol, Marius; Aqeilan, Rami I.; Nawaz, Zafar; Farooq, Amjad

2012-01-01

The WWOX tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically-relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically-distinct E66/Y85 duo at structurally-equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, introduction of E66R/Y85W double-substitution within the WW2 domain not only results in gain-of-function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease. PMID:22634283

Molecular origin of the binding of WWOX tumor suppressor to ErbB4 receptor tyrosine kinase.

PubMed

Schuchardt, Brett J; Bhat, Vikas; Mikles, David C; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

2013-12-23

The ability of WWOX tumor suppressor to physically associate with the intracellular domain (ICD) of ErbB4 receptor tyrosine kinase is believed to play a central role in downregulating the transcriptional function of the latter. Herein, using various biophysical methods, we show that while the WW1 domain of WWOX binds to PPXY motifs located within the ICD of ErbB4 in a physiologically relevant manner, the WW2 domain does not. Importantly, while the WW1 domain absolutely requires the integrity of the PPXY consensus sequence, nonconsensus residues within and flanking this motif do not appear to be critical for binding. This strongly suggests that the WW1 domain of WWOX is rather promiscuous toward its cellular partners. We also provide evidence that the lack of binding of the WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Consistent with this notion, the Y85W substitution within the WW2 domain exquisitely restores its binding to PPXY motifs in a manner akin to the binding of the WW1 domain of WWOX. Of particular significance is the observation that the WW2 domain augments the binding of the WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1-WW2 tandem module of WWOX in agreement with our findings reported previously. Altogether, our study sheds new light on the molecular basis of an important WW-ligand interaction involved in mediating a plethora of cellular processes.

Molecular Origin of the Binding of WWOX Tumor Suppressor to ErbB4 Receptor Tyrosine Kinase

PubMed Central

Schuchardt, Brett J.; Bhat, Vikas; Mikles, David C.; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

2014-01-01

The ability of WWOX tumor suppressor to physically associate with the intracellular domain (ICD) of ErbB4 receptor tyrosine kinase is believed to play a central role in down-regulating the transcriptional function of the latter. Herein, using various biophysical methods, we show that while the WW1 domain of WWOX binds to PPXY motifs located within the ICD of ErbB4 in a physiologically-relevant manner, the WW2 domain does not. Importantly, while the WW1 domain absolutely requires the integrity of the PPXY consensus sequence, non-consensus residues within and flanking this motif do not appear to be critical for binding. This strongly suggests that the WW1 domain of WWOX is rather promiscuous toward its cellular partners. We also provide evidence that the lack of binding of WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Consistent with this notion, the Y85W substitution within the WW2 domain exquisitely restores its binding to PPXY motifs in a manner akin to the binding of WW1 domain of WWOX. Of particular significance is the observation that WW2 domain augments the binding of WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1–WW2 tandem module of WWOX in agreement with our findings reported previously. Taken together, our study sheds new light on the molecular basis of an important WW-ligand interaction involved in mediating a plethora of cellular processes. PMID:24308844

Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III.

PubMed

Ahmad, Basir; Ahmed, Md Zulfazal; Haq, Soghra Khatun; Khan, Rizwan Hasan

2005-06-15

The effect of guanidine hydrochloride (GnHCl) on the global stability of human serum albumin (HSA) has been studied by fluorescence and circular dichroism spectroscopic measurements. The differential stability of native conformation of three HSA domains were explored by using domain-specific ligands, hemin (domain I), chloroform (domain II), bilirubin (at domain I/domain II interface) and diazepam (domain III). GnHCl induced unfolding transition curves as monitored by probes for secondary and tertiary structures were cooperative but noncoincidental. A strong ANS binding to the protein was observed around 1.8 M GnHCl, suggesting existence of intermediate states in the unfolding pathway of HSA. A gradual decrease (in the GnHCl concentration range 0.0-1.8 M) in the binding of diazepam indicates that domain III is the most labile to GnHCl denaturation. A significant increase in the binding of bilirubin up to 1.4 M GnHCl and decrease thereafter leading to complete abolishment of bilirubin binding at around 2.0 M GnHCl suggest favorable rearrangement and separation of domains I and II at 1.4 and 2.0 M GnHCl concentration, respectively. Above 1.6 M GnHCl, decrease of the binding of hemin, a ligand for domain I, chloroform, which binds in domain II and lone tryptophanyl fluorescence (Trp-214 located in domain II) indicate that at higher concentration of GnHCl domains I and II start unfolding simultaneously but the stability of domain I (7.4 Kcal/mol) is much more than domain II (4.3 Kcal/mol). A pictorial model for the unfolding of HSA domains, consistent with all these results, has been formulated, suggesting that domain III is the most labile followed by domain II while domain I is the most stable. A molten globule like state of domain III around 1.8 M GnHCl has also been identified and characterized.

Molecular Basis for Failure of “Atypical” C1 Domain of Vav1 to Bind Diacylglycerol/Phorbol Ester*

PubMed Central

Geczy, Tamas; Peach, Megan L.; El Kazzouli, Saïd; Sigano, Dina M.; Kang, Ji-Hye; Valle, Christopher J.; Selezneva, Julia; Woo, Wonhee; Kedei, Noemi; Lewin, Nancy E.; Garfield, Susan H.; Lim, Langston; Mannan, Poonam; Marquez, Victor E.; Blumberg, Peter M.

2012-01-01

C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu9, Glu10, Thr11, Thr24, and Tyr26) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1. PMID:22351766

A Method for Measuring Fishing Effort by Small-Scale Fish Aggregating Device (FAD) Fishers from the Commonwealth of Dominica

ERIC Educational Resources Information Center

Alvard, Michael; McGaffey, Ethan; Carlson, David

2015-01-01

We used global positioning system (GPS) technology and tracking analysis to measure fishing effort by marine, small-scale, fish aggregating device (FAD) fishers of the Commonwealth of Dominica. FADs are human-made structures designed to float on the surface of the water and attract fish. They are also prone to common pool resource problems. To…

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of Acidic Fibroblast Growth Factor

PubMed Central

Jayanthi, Srinivas; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy; Furr, Mercede; Daily, Anna; Thurman, Ryan; Rutherford, Lindsay; Chandrashekar, Reena; Adams, Paul; Prudovsky, Igor; Suresh Kumar, Thallapuranam Krishnaswamy

2014-01-01

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER) -Golgi secretory pathway. FGF1 is released through a Cu2+ - mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu2+- mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that binding of Cu2+ to the C2B domain is important for the release of FGF1 in to the extracellular medium. In this study, using a variety of biophysical studies, Cu2+ and lipid interactions of the C2B domain of Syt1were characterized. Isothermal titration calorimetry (ITC) experiments reveal that C2B domain binds to Cu2+ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb3+ show that there are two Cu2+- binding pockets on the C2B domain, and one of these is also a Ca2+- binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu2+. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1. PMID:25224745

[Development of free will and determinism scale in Japanese].

PubMed

Goto, Takayuki; Ishibashi, Yuya; Kajimura, Shogo; Oka, Ryunosuke; Kusumi, Takashi

2015-04-01

We developed a free will and determinism scale in Japanese (FAD-J) to assess lay beliefs in free will, scientific determinism, fatalistic determinism, and unpredictability. In Study 1, we translated a free will and determinism scale (FAD-Plus) into Japanese and verified its reliability and validity. In Study 2, we examined the relationship between the FAD-J and eight other scales. Results suggested that lay beliefs in free will and determinism were related to self-regulation, critical thinking, other-oriented empathy, self-esteem, and regret and maximization in decision makings. We discuss the usefulness of the FAD-J for studying the psychological functions of lay beliefs in free will and determinism.

Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

PubMed

Bussiere, D E; Kong, X; Egan, D A; Walter, K; Holzman, T F; Lindh, F; Robins, T; Giranda, V L

1998-11-01

The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

PubMed Central

Cooper, J A; Kashishian, A

1993-01-01

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins.

PubMed

Raman, Rajeev; Rajanikanth, V; Palaniappan, Raghavan U M; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P; Sharma, Yogendra; Chang, Yung-Fu

2010-12-29

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th) (Lig A9) and 10(th) repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins

PubMed Central

Palaniappan, Raghavan U. M.; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P.; Sharma, Yogendra; Chang, Yung-Fu

2010-01-01

Background Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. Principal Findings We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. Conclusions We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding. PMID:21206924

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

The ATPase domain of the large terminase protein, gp17, from bacteriophage T4 binds DNA: implications to the DNA packaging mechanism.

PubMed

Alam, Tanfis I; Rao, Venigalla B

2008-03-07

Translocation of double-stranded DNA into a preformed capsid by tailed bacteriophages is driven by powerful motors assembled at the special portal vertex. The motor is thought to drive processive cycles of DNA binding, movement, and release to package the viral genome. In phage T4, there is evidence that the large terminase protein, gene product 17 (gp17), assembles into a multisubunit motor and translocates DNA by an inchworm mechanism. gp17 consists of two domains; an N-terminal ATPase domain (amino acids 1-360) that powers translocation of DNA, and a C-terminal nuclease domain (amino acids 361-610) that cuts concatemeric DNA to generate a headful-size viral genome. While the functional motifs of ATPase and nuclease have been well defined and the ATPase atomic structure has been solved, the DNA binding motif(s) responsible for viral DNA recognition, cutting, and translocation are unknown. Here we report the first evidence for the presence of a double-stranded DNA binding activity in the gp17 ATPase domain. Binding to DNA is sensitive to Mg(2+) and salt, but not the type of DNA used. DNA fragments as short as 20 bp can bind to the ATPase but preferential binding was observed to DNA greater than 1 kb. A high molecular weight ATPase-DNA complex was isolated by gel filtration, suggesting oligomerization of ATPase following DNA interaction. DNA binding was not observed with the full-length gp17, or the C-terminal nuclease domain. The small terminase protein, gp16, inhibited DNA binding, which was further accentuated by ATP. The presence of a DNA binding site in the ATPase domain and its binding properties implicate a role in the DNA packaging mechanism.

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding

PubMed Central

Kostrhon, Sebastian; Kontaxis, Georg; Kaufmann, Tanja; Schirghuber, Erika; Kubicek, Stefan; Konrat, Robert

2017-01-01

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry. PMID:28864776

DISTINCT ROLES OF β1 MIDAS, ADMIDAS AND LIMBS CATION-BINDING SITES IN LIGAND RECOGNITION BY INTEGRIN α2β1*

PubMed Central

Valdramidou, Dimitra; Humphries, Martin J.; Mould, A. Paul

2012-01-01

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS) and LIMBS (ligand-associated metal binding site). The role of these sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating mAb TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+ and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding site, occupancy of which increases the affinity of Mg2+ for the MIDAS. PMID:18820259

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

PubMed

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Identification and Analysis of a Gene from Calendula officinalis Encoding a Fatty Acid Conjugase

PubMed Central

Qiu, Xiao; Reed, Darwin W.; Hong, Haiping; MacKenzie, Samuel L.; Covello, Patrick S.

2001-01-01

Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Δ12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes. PMID:11161042

Application of a flush airdata sensing system to a wing leading edge (LE-FADS)

NASA Technical Reports Server (NTRS)

Whitmore, Stephen A.; Moes, Timothy R.; Czerniejewski, Mark W.; Nichols, Douglas A.

1993-01-01

This paper investigates the feasibility of locating a flush air-data sensing (FADS) system on a wing leading edge where the operation of the avionics or fire control radar system will not be hindered. The leading-edge FADS system (LE-FADS) was installed on an unswept symmetrical airfoil, and a series of low-speed wind-tunnel tests were conducted to evaluate the performance of the system. As a result of the tests it is concluded that the aerodynamic models formulated for use on aircraft nosetips are directly applicable to wing leading edges and that the calibration process is similar. Furthermore, the agreement between the air-data calculations for angle of attack and total pressure from the LE-FADS and known wind-tunnel values suggest that wing-based flush air-data systems can be calibrated to a high degree of accuracy. Static wind-tunnel tests for angles of attack from -50 to 50 deg and dynamic pressures from 3.6 to 11.4 lb/sq ft were performed.

Application of a Flush Airdata Sensing System to a Wing Leading Edge (LE-FADS)

NASA Technical Reports Server (NTRS)

Whitmore, Stephen A.; Moes, Timothy R.; Czerniejewski, Mark W.; Nichols, Douglas A.

1993-01-01

The feasibility of locating a flush airdata sensing (FADS) system on a wing leading edge where the operation of the avionics or fire control radar system will not be hindered is investigated. The leading-edge FADS system (LE-FADS) was installed on an unswept symmetrical airfoil and a series of low-speed wind-tunnel tests were conducted to evaluate the performance of the system. As a result of the tests it is concluded that the aerodynamic models formulated for use on aircraft nosetips are directly applicable to wing leading edges and that the calibration process is similar. Furthermore, the agreement between the airdata calculations for angle of attack and total pressure from the LE-FADS and known wind-tunnel values suggest that wing-based flush airdata systems can be calibrated to a high degree of accuracy. Static wind-tunnel tests for angles of attack from -50 deg to 50 deg and dynamic pressures from 3.6 to 11.4 lb/sq ft were performed.

Immobilization of flavin adenine dinucleotide (FAD) onto carbon cloth and its application as working electrode in an electroenzymatic bioreactor.

PubMed

Jayabalan, R; Sathishkumar, M; Jeong, E S; Mun, S P; Yun, S E

2012-11-01

A high porosity carbon cloth with immobilized FAD was employed as working electrode in electrochemical NADH-regeneration procedure. Carbon cloth was oxidized with hot acids to create surface carboxyl group and then coupled by adenine amino group of FAD with carbodiimide in the presence of N-hydroxysulfosuccinimide. The bioelectrocatalytic NADH-regeneration was coupled to the conversion of achiral substrate pyruvate into chiral product l-lactate by l-lactate dehydrogenase (l-LDH) within the same reactor. The conversion was completed at 96h in bioreactor with FAD-modified carbon cloth, resulting in about 6mM of l-lactate from 10mM of pyruvate. While with bare carbon cloth, the yield at 120h was around 5mM. Immobilized FAD on the surface of carbon cloth electrode facilitated it to carry electrons from electrode to electron transfer enzymes; thereby NADH-regeneration was accelerated to drive the enzymatic reaction efficiently. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dietary adaptation of FADS genes in Europe varied across time and geography.

PubMed

Ye, Kaixiong; Gao, Feng; Wang, David; Bar-Yosef, Ofer; Keinan, Alon

2017-05-26

Fatty acid desaturase (FADS) genes encode rate-limiting enzymes for the biosynthesis of omega-6 and omega-3 long-chain polyunsaturated fatty acids (LCPUFAs). This biosynthesis is essential for individuals subsisting on LCPUFA-poor diets (for example, plant-based). Positive selection on FADS genes has been reported in multiple populations, but its cause and pattern in Europeans remain unknown. Here we demonstrate, using ancient and modern DNA, that positive selection acted on the same FADS variants both before and after the advent of farming in Europe, but on opposite (that is, alternative) alleles. Recent selection in farmers also varied geographically, with the strongest signal in southern Europe. These varying selection patterns concur with anthropological evidence of varying diets, and with the association of farming-adaptive alleles with higher FADS1 expression and thus enhanced LCPUFA biosynthesis. Genome-wide association studies reveal that farming-adaptive alleles not only increase LCPUFAs, but also affect other lipid levels and protect against several inflammatory diseases.

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

PubMed Central

Budhidarmo, Rhesa; Day, Catherine L.

2014-01-01

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturatingmore » conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.« less

Hydropericardium Hepatitis Syndrome Emerged in Cherry Valley Ducks in China.

PubMed

Chen, H; Dou, Y; Zheng, X; Tang, Y; Zhang, M; Zhang, Y; Wang, Z; Diao, Y

2017-08-01

Since June 2015, a highly pathogenic disease occurred in duck flocks in China, causing pericardial effusion, enlarged discoloured liver, renal enlargement and haemorrhagic lung with a mortality ranging from 5% to 20%. Previous study confirmed that Fowl adenovirus group C (FAdV-C) and some field FAdVs isolates had been identified as causative agents of hydropericardium hepatitis syndrome (HHS) in chickens and geese world widely. In this study, we firstly report the isolation of FAdV-C from ducks with HHS. The two isolates, designated as SDSX and SDJX, were separated from liver samples using 9-day-old SPF chicken embryos and could cause severe cytopathic effects in duck and chicken embryonic kidney cells. The entire ORF sequences of hexon gene of the two isolates were amplified, sequenced and analysed by restriction fragment length polymorphism. Phylogenetic analysis of loop 1 sequences of hexon gene of FAdVs revealed that the two isolates were closely related to FAdV-C isolates, which could cause HHS in chickens. Experimental infection indicated that the isolate was high pathogenicity to 20-day-old ducks. Our study shows that the recently emerged HHS in ducks was caused by FAdV-C and may possess a potential risk to other poultry flocks. © 2016 Blackwell Verlag GmbH.

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds.

PubMed

Rolletschek, Hardy; Borisjuk, Ljudmilla; Sánchez-García, Alicia; Gotor, Cecilia; Romero, Luis C; Martínez-Rivas, José M; Mancha, Manuel

2007-01-01

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.

Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens.

PubMed

Dar, Arshud; Gomis, Susantha; Shirley, Ian; Mutwiri, George; Brownlie, Robert; Potter, Andrew; Gerdts, Volker; Tikoo, Suresh K

2012-03-01

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.

Structural analysis of fungus-derived FAD glucose dehydrogenase

PubMed Central

Yoshida, Hiromi; Sakai, Genki; Mori, Kazushige; Kojima, Katsuhiro; Kamitori, Shigehiro; Sode, Koji

2015-01-01

We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management. PMID:26311535

Histone acetyltransferase general control non-repressed protein 5 (GCN5) affects the fatty acid composition of Arabidopsis thaliana seeds by acetylating fatty acid desaturase3 (FAD3).

PubMed

Wang, Tianya; Xing, Jiewen; Liu, Xinye; Liu, Zhenshan; Yao, Yingyin; Hu, Zhaorong; Peng, Huiru; Xin, Mingming; Zhou, Dao-Xiu; Zhang, Yirong; Ni, Zhongfu

2016-12-01

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α-linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA-Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIβ as the targets of GCN5. Notably, the GCN5-dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild-type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

High-intensity focused ultrasound for the treatment of fibroadenomata (HIFU-F) study.

PubMed

Peek, Mirjam C L; Ahmed, Muneer; Douek, Michael

2015-01-01

Breast fibroadenomata (FAD) are the most common benign lesions in women. For palpable lesions, there are currently three standard treatment options: reassurance (with or without follow-up), vacuum-assisted mammotomy (VAM) or surgical excision. High-intensity focused ultrasound (HIFU) ablation has been used in the treatment of FAD. The drawback of HIFU is its prolonged treatment duration. The aim of this trial is to evaluate circumferential HIFU treatment for the effective ablation of FAD with a reduced treatment time. Fifty patients (age ≥18 years) will be recruited with symptomatic FAD, visible on ultrasound (US, grade U2 benign). In patients ≥25 years, cytology or histology will be performed to confirm the diagnosis of a FAD. These patients will receive HIFU treatment using the US-guided Echopulse device (Theraclion Ltd., Malakoff, France) under local anaesthesia. An additional 50 patients will be recruited and contacted 6 months after discharge from the breast clinic. These patients will be offered an US scan to determine the change in size of their FAD. This natural change in size will be compared to the decrease in size after HIFU treatment. Secondary outcome measures include post-treatment complications, patient recorded outcome measures, mean treatment time and cost analysis. Current Controlled Trials: ISRCTN76622747.

The evaluation of family functioning by the family assessment device: a systematic review of studies in adult clinical populations.

PubMed

Staccini, Laura; Tomba, Elena; Grandi, Silvana; Keitner, Gabor I

2015-03-01

A large body of research, documenting the impact of a family's functioning on health outcomes, highlights the importance of introducing the evaluation of patients' family dynamics into clinical judgment. The Family Assessment Device (FAD) is a self-report questionnaire designed to assess specific dimensions of family functioning. This qualitative systematic review, which follows PRISMA guidelines, aimed to identify the FAD's clinimetric properties and to report the incremental utility of its inclusion in clinical settings. A thorough literature search was performed, using both computerized and manual searches, yielding a total of 148 studies that were included in this review. The FAD has been extensively used in a variety of research contexts. In the majority of studies it was able to discriminate between clinical populations and controls and among groups of patients with different illnesses. The FAD also showed good test-retest and concurrent reliability, and modest sensitivity to change after treatment. FAD-dysfunctional family functioning was related to several patient clinical outcomes, including lower recovery rates and adherence to treatment, longer recovery time, poorer quality of life, and increased risk of relapse and drop-out. The present review demonstrates that the FAD is a suitable instrument for the evaluation of family functioning both in clinical and research settings. © 2014 Family Process Institute.

Electrocatalytic reaction of hydrogen peroxide and NADH based on poly(neutral red) and FAD hybrid film.

PubMed

Lin, Kuo Chiang; Lin, Yu Ching; Chen, Shen Ming

2012-01-07

A simple method to immobilize poly(neutral red) (PNR) and flavin adenine dinucleotide (FAD) hybrid film (PNR/FAD) by cyclic voltammetry is proposed. The PNR/FAD hybrid film can be easily prepared on an electrode surface involving electropolymerization of neutral red (NR) monomers and the electrostatic interaction between the positively charged PNR and the negatively charged FAD. It exhibits electroactive, stable, surface-confined, pH-dependent, nano-sized, and compatible properties. It provides good electrocatalytic properties to various species. It shows a sensitivity of 5.4 μA mM(-1) cm(-2) and 21.5 μA mM(-1) cm(-2) for hydrogen peroxide (H(2)O(2)) and nicotinamide adenine dinucleotide (NADH) with the linear range of 0.1 μM-39 mM and 5 × 10(-5) to 2.5 × 10(-4) M, respectively. It shows another linear range of 48.8-355.5 mM with the sensitivity of 12.3 μA mM(-1) cm(-2) for H(2)O(2). In particular, the PNR/FAD hybrid film has potential to replace some hemoproteins to be a cathode of biofuel cells and provide the biosensing system for glucose and ethanol. This journal is © The Royal Society of Chemistry 2012

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.

PubMed

Choudhury, Nila Roy; Heikel, Gregory; Trubitsyna, Maryia; Kubik, Peter; Nowak, Jakub Stanislaw; Webb, Shaun; Granneman, Sander; Spanos, Christos; Rappsilber, Juri; Castello, Alfredo; Michlewski, Gracjan

2017-11-08

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.

GPU-Based Point Cloud Superpositioning for Structural Comparisons of Protein Binding Sites.

PubMed

Leinweber, Matthias; Fober, Thomas; Freisleben, Bernd

2018-01-01

In this paper, we present a novel approach to solve the labeled point cloud superpositioning problem for performing structural comparisons of protein binding sites. The solution is based on a parallel evolution strategy that operates on large populations and runs on GPU hardware. The proposed evolution strategy reduces the likelihood of getting stuck in a local optimum of the multimodal real-valued optimization problem represented by labeled point cloud superpositioning. The performance of the GPU-based parallel evolution strategy is compared to a previously proposed CPU-based sequential approach for labeled point cloud superpositioning, indicating that the GPU-based parallel evolution strategy leads to qualitatively better results and significantly shorter runtimes, with speed improvements of up to a factor of 1,500 for large populations. Binary classification tests based on the ATP, NADH, and FAD protein subsets of CavBase, a database containing putative binding sites, show average classification rate improvements from about 92 percent (CPU) to 96 percent (GPU). Further experiments indicate that the proposed GPU-based labeled point cloud superpositioning approach can be superior to traditional protein comparison approaches based on sequence alignments.

Serum Lipid Concentrations and FADS Genetic Variants in Young Mexican College Students: The UP-AMIGOS Cohort Study.

PubMed

Vazquez-Vidal, Itzel; Voruganti, V Saroja; Hannon, Bridget A; Andrade, Flavia Cristina Drumond; Aradillas-García, Celia; Nakamura, Manabu T; Terán-García, Margarita

2018-05-30

Recent genome-wide association studies in the Mexican population have identified several genetic loci associated with blood lipid levels in adults. However, studies focusing on the fatty acid desaturase (FADS) gene cluster have been understudied in this population, even though it seems associated with lipid profiles in other ethnicities. The aim of this study was to test associations between single nucleotide polymorphisms (SNPs) in the FADS cluster (rs174546, rs1535, rs174548, rs174550, rs174450, and rs174618) and serum lipid profiles in young Mexicans. Anthropometrics, serum lipid profiles, and FADS SNPs were measured in 998 subjects in the UP-AMIGOS cohort study. Genotype-phenotype (total cholesterol [TC], triglyceride [TG], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], and very-low-density lipoprotein [VLDL]) associations were assessed using PLINK adjusted for sex, age, and body mass index (BMI). Among 6 FADS SNPs, we found that carriers of the C-allele of the FADS1-rs174546 showed a significant association with lower TG concentrations (β = -12.6 mg/dL, p = 0.009) and lower VLDL concentrations (β = -2.52 mg/dL, p = 0.005). We found that rs174546, rs1535, and rs174550 were in high linkage disequilibrium (r2 > 0.80). There were no significant associations between rs174550, rs174548, and rs174618 and lipid profiles. A genetic variant in the FADS1 (rs174546) gene is a major contributor of plasma TG and VLDL concentrations in healthy young Mexicans. © 2018 S. Karger AG, Basel.

Measles virus fusion machinery activated by sialic acid binding globular domain.

PubMed

Talekar, Aparna; Moscona, Anne; Porotto, Matteo

2013-12-01

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Molecular Evolution of the Oxygen-Binding Hemerythrin Domain

PubMed Central

Alvarez-Carreño, Claudia; Becerra, Arturo; Lazcano, Antonio

2016-01-01

Background The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins) are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes. Results Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid) cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role. Conclusions Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later evolution of the oxygen-binding hemerythrin domain in both prokaryotes and eukaryotes led to a wide variety of functions, ranging from protection against oxidative damage in anaerobic and microaerophilic organisms, to oxygen supplying to particular enzymes and pathways in aerobic and facultative species. PMID:27336621

Characterizing protein domain associations by Small-molecule ligand binding

PubMed Central

Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

2012-01-01

Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

Allostery Mediates Ligand Binding to WWOX Tumor Suppressor via a Conformational Switch

PubMed Central

Schuchardt, Brett J.; Mikles, David C.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

2014-01-01

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to WW1 domain in the context of WW1-WW2 tandem module of WWOX tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of WW1-WW2 tandem module to an array of putative PPXY ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically-relevant manner, the WW2 domain does not. Moreover, ligand binding to WW1 domain in the context of WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of WW2 domain with WW1 blocks access to ligand. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of WW2 orphan domain to chaperone physiological action of WW1 domain within the context of the WW1-WW2 tandem module of WWOX. PMID:25703206

Loss of Function of FATTY ACID DESATURASE7 in Tomato Enhances Basal Aphid Resistance in a Salicylate-Dependent Manner1[W][OA

PubMed Central

Avila, Carlos A.; Arévalo-Soliz, Lirio M.; Jia, Lingling; Navarre, Duroy A.; Chen, Zhaorigetu; Howe, Gregg A.; Meng, Qing-Wei; Smith, Jonathon E.; Goggin, Fiona L.

2012-01-01

We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families. PMID:22291202

Chromosome 14 and late-onset familial alzheimer disease (FAD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schellenberg, G.D.; Anderson, L.; Nemens, E.

1993-09-01

Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analysis were used, in 49 families with a mean age at onset [>=]60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependentmore » penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score [>=]2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P<.02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52, however, was found for a subgroup of families of intermediate age at onset (mean age at onset [>=]60 years and <70 years). These results indicate that the chromosome 14 locus is not responsible for Alzheimer disease in most late-onset FAD kindreds but could play a role in a subset of these kindreds. 37 refs., 1 fig., 6 tabs.« less

A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74.

PubMed

Bosselut, R; Levin, J; Adjadj, E; Ghysdael, J

1993-11-11

Ets proteins form a family of sequence specific DNA binding proteins which bind DNA through a 85 aminoacids conserved domain, the Ets domain, whose sequence is unrelated to any other characterized DNA binding domain. Unlike all other known Ets proteins, which bind specific DNA sequences centered over either GGAA or GGAT core motifs, E74 and Elf1 selectively bind to GGAA corecontaining sites. Elf1 and E74 differ from other Ets proteins in three residues located in an otherwise highly conserved region of the Ets domain, referred to as conserved region III (CRIII). We show that a restricted selectivity for GGAA core-containing sites could be conferred to Ets1 upon changing a single lysine residue within CRIII to the threonine found in Elf1 and E74 at this position. Conversely, the reciprocal mutation in Elf1 confers to this protein the ability to bind to GGAT core containing EBS. This, together with the fact that mutation of two invariant arginine residues in CRIII abolishes DNA binding, indicates that CRIII plays a key role in Ets domain recognition of the GGAA/T core motif and lead us to discuss a model of Ets proteins--core motif interaction.

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

PubMed

Scheele, Urte; Alves, Jurgen; Frank, Ronald; Duwel, Michael; Kalthoff, Christoph; Ungewickell, Ernst

2003-07-11

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, R.; Wilton, R.; Cuff, M. E.

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes butmore » have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.« less

The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lei; Zhang, Qing; Yang, Yu

Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required formore » RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.« less

Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex*

PubMed Central

Das, Devashish; Faridounnia, Maryam; Kovacic, Lidija; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E.

2017-01-01

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble. PMID:28028171

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

PubMed Central

Hanaoka, Shingo; Nagadoi, Aritaka; Nishimura, Yoshifumi

2005-01-01

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2. PMID:15608118

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PubMed

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes.

PubMed

Matern, Andreas; Pedrolli, Danielle; Großhennig, Stephanie; Johansson, Jörgen; Mack, Matthias

2016-12-01

The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B 2 ), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Cooperative Allosteric Ligand Binding in Calmodulin

NASA Astrophysics Data System (ADS)

Nandigrami, Prithviraj

Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (micro). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of micro. First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM. Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops. Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM. (Abstract shortened by ProQuest.).

Mapping small molecule binding data to structural domains

PubMed Central

2012-01-01

Background Large-scale bioactivity/SAR Open Data has recently become available, and this has allowed new analyses and approaches to be developed to help address the productivity and translational gaps of current drug discovery. One of the current limitations of these data is the relative sparsity of reported interactions per protein target, and complexities in establishing clear relationships between bioactivity and targets using bioinformatics tools. We detail in this paper the indexing of targets by the structural domains that bind (or are likely to bind) the ligand within a full-length protein. Specifically, we present a simple heuristic to map small molecule binding to Pfam domains. This profiling can be applied to all proteins within a genome to give some indications of the potential pharmacological modulation and regulation of all proteins. Results In this implementation of our heuristic, ligand binding to protein targets from the ChEMBL database was mapped to structural domains as defined by profiles contained within the Pfam-A database. Our mapping suggests that the majority of assay targets within the current version of the ChEMBL database bind ligands through a small number of highly prevalent domains, and conversely the majority of Pfam domains sampled by our data play no currently established role in ligand binding. Validation studies, carried out firstly against Uniprot entries with expert binding-site annotation and secondly against entries in the wwPDB repository of crystallographic protein structures, demonstrate that our simple heuristic maps ligand binding to the correct domain in about 90 percent of all assessed cases. Using the mappings obtained with our heuristic, we have assembled ligand sets associated with each Pfam domain. Conclusions Small molecule binding has been mapped to Pfam-A domains of protein targets in the ChEMBL bioactivity database. The result of this mapping is an enriched annotation of small molecule bioactivity data and a grouping of activity classes following the Pfam-A specifications of protein domains. This is valuable for data-focused approaches in drug discovery, for example when extrapolating potential targets of a small molecule with known activity against one or few targets, or in the assessment of a potential target for drug discovery or screening studies. PMID:23282026

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Han; Shin, Ok-Ho; Machius, Mischa

The neuronal protein synaptotagmin 1 functions as a Ca{sup 2+} sensor in exocytosis via two Ca{sup 2+}-binding C{sub 2} domains. The very similar synaptotagmin 4, which includes all the predicted Ca{sup 2+}-binding residues in the C{sub 2}B domain but not in the C{sub 2}A domain, is also thought to function as a neuronal Ca{sup 2+} sensor. Here we show that, unexpectedly, both C{sub 2} domains of fly synaptotagmin 4 exhibit Ca{sup 2+}-dependent phospholipid binding, whereas neither C{sub 2} domain of rat synaptotagmin 4 binds Ca{sup 2+} or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca{sup 2+}more » ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C{sub 2}B domain unable to form full Ca{sup 2+}-binding sites. These results indicate that synaptotagmin 4 is a Ca{sup 2+} sensor in the fly but not in the rat, that the Ca{sup 2+}-binding properties of C{sub 2} domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.« less

A high-resolution structure of the DNA-binding domain of AhrC, the arginine repressor/activator protein from Bacillus subtilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The structure of the winged helix–turn–helix DNA-binding domain of AhrC has been determined at 1.0 Å resolution. The largely hydrophobic β-wing shows high B factors and may mediate the dimer interface in operator complexes. In Bacillus subtilis the concentration of l-arginine is controlled by the transcriptional regulator AhrC, which interacts with 18 bp DNA operator sites called ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a 100 kDa homohexamer, with each subunit having two domains. The C-terminal domains form the core, mediating intersubunit interactions and binding of the co-repressor l-arginine, whilst the N-terminal domains containmore » a winged helix–turn–helix DNA-binding motif and are arranged around the periphery. The N-terminal domain of AhrC has been expressed, purified and characterized and it has been shown that the fragment still binds DNA operators as a recombinant monomer. The DNA-binding domain has also been crystallized and the crystal structure refined to 1.0 Å resolution is presented.« less

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping

PubMed Central

Stähle, I; Brizzio, C; Barile, M; Brandsch, R

1999-01-01

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (αFp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by αFp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to αFp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and αFP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-d-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize αFp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-d-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the αFp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by αFp-AB comprises, besides the flavin moiety, protein secondary structure elements. PMID:10193410

Absorption and emission spectroscopic characterization of blue-light receptor Slr1694 from Synechocystis sp. PCC6803.

PubMed

Zirak, P; Penzkofer, A; Lehmpfuhl, C; Mathes, T; Hegemann, P

2007-01-03

The BLUF protein Slr1694 from the cyanobacterium Synechocystis sp. PCC6803 is characterized by absorption and emission spectroscopy. Slr1694 expressed from E. coli which non-covalently binds FAD, FMN, and riboflavin (called Slr1694(I)), and reconstituted Slr1694 which dominantly contains FAD (called Slr1694(II)) are investigated. The receptor conformation of Slr1694 (dark adapted form Slr1694(r)) is transformed to the putative signalling state (light adapted form Slr1694(s)) with red-shifted absorption and decreased fluorescence efficiency by blue-light excitation. In the dark at 22 degrees C, the signalling state recovers back to the initial receptor state with a time constants of about 14.2s for Slr1694(I) and 17s for Slr1694(II). Quantum yields of signalling state formation of approximately 0.63+/-0.07 for both Slr1694(I) and Slr1694(II) were determined by transient transmission measurements and intensity dependent steady-state transmission measurements. Extended blue-light excitation causes some bound flavin conversion to the hydroquinone form and some photo-degradation, both with low quantum efficiency. The flavin-hydroquinone re-oxidizes slowly back (time constant 5-9 min) to the initial flavoquinone form in the dark. A photo-cycle dynamics scheme is presented.

Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607

NASA Astrophysics Data System (ADS)

Schiering, N.; Kabsch, W.; Moore, M. J.; Distefano, M. D.; Walsh, C. T.; Pai, E. F.

1991-07-01

SEVERAL hundred million tons of toxic mercurials are dispersed in the biosphere1. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase2 and mercuric ion reductase (MerA) 3-5. The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases6, catalyses the reaction NADPH + Hg(II) --> NADP+ + H+Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase7,8 serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure9 and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), pI258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved10,11. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn5Ol and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon11. These domains can be proteolytically cleaved off without changing the catalytic efficiency3. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

PubMed Central

VanScyoc, Wendy S; Sorensen, Brenda R; Rusinova, Elena; Laws, William R; Ross, J B Alexander; Shea, Madeline A

2002-01-01

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition. PMID:12414709

Signatures of Pleiotropy, Economy and Convergent Evolution in a Domain-Resolved Map of Human–Virus Protein–Protein Interaction Networks

PubMed Central

Garamszegi, Sara; Franzosa, Eric A.; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are otherwise hidden in the traditional binary network, highlighting the power and necessity of high-resolution approaches in host-pathogen systems biology. PMID:24339775

Signatures of pleiotropy, economy and convergent evolution in a domain-resolved map of human-virus protein-protein interaction networks.

PubMed

Garamszegi, Sara; Franzosa, Eric A; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are otherwise hidden in the traditional binary network, highlighting the power and necessity of high-resolution approaches in host-pathogen systems biology.

Biological effects of individually synthesized TNF-binding domain of variola virus CrmB protein.

PubMed

Tsyrendorzhiev, D D; Orlovskaya, I A; Sennikov, S V; Tregubchak, T V; Gileva, I P; Tsyrendorzhieva, M D; Shchelkunov, S N

2014-06-01

The biological characteristics of a 17-kDa protein synthesized in bacterial cells, a TNF-binding domain (VARV-TNF-BP) of a 47-kDa variola virus CrmB protein (VARV-CrmB) consisting of TNF-binding and chemokine-binding domains, were studied. Removal of the C-terminal chemokine-binding domain from VARV-CrmB protein was inessential for the efficiency of its inhibition of TNF cytotoxicity towards L929 mouse fibroblast culture and for TNF-induced oxidative metabolic activity of mouse blood leukocytes. The results of this study could form the basis for further studies of VARV-TNF-BP mechanisms of activity for prospective use in practical medicine.

Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch.

PubMed

Schuchardt, Brett J; Mikles, David C; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

2015-04-01

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1-WW2 tandem module of WW domain-containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1-WW2 tandem module to an array of putative proline-proline-x-tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1-WW2 tandem module of WWOX. Copyright © 2015 John Wiley & Sons, Ltd.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

The regulation of integrin function by divalent cations

PubMed Central

Zhang, Kun; Chen, JianFeng

2012-01-01

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca2+-binding motifs that have essential roles in integrin biogenesis. The function of another Ca2+-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions. PMID:22647937

AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy

PubMed Central

Spindler, Matthew J.; Burmeister, Brian T.; Huang, Yu; Hsiao, Edward C.; Salomonis, Nathan; Scott, Mark J.; Srivastava, Deepak; Carnegie, Graeme K.; Conklin, Bruce R.

2013-01-01

Background A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown. Methodology/Principal Findings To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction. Conclusions These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy. PMID:23658642

Experimentally biased model structure of the Hsc70/auxilin complex: Substrate transfer and interdomain structural change

PubMed Central

Gruschus, James M.; Greene, Lois E.; Eisenberg, Evan; Ferretti, James A.

2004-01-01

A model structure of the Hsc70/auxilin complex has been constructed to gain insight into interprotein substrate transfer and ATP hydrolysis induced conformational changes in the multidomain Hsc70 structure. The Hsc70/auxilin system, which is a member of the Hsp70/Hsp40 chaperone system family, uncoats clathrin-coated vesicles in an ATP hydrolysis-driven process. Incorporating previous results from NMR and mutant binding studies, the auxilin J-domain was docked into the Hsc70 ATPase domain lower cleft using rigid backbone/flexible side chain molecular dynamics, and the Hsc70 substrate binding domain was docked by a similar procedure. For comparison, J-domain and substrate binding domain docking sites were obtained by the rigid-body docking programs DOT and ZDOCK, filtered and ranked by the program ClusPro, and relaxed using the same rigid backbone/flexible side chain dynamics. The substrate binding domain sites were assessed in terms of conserved surface complementarity and feasibility in the context of substrate transfer, both for auxilin and another Hsp40 protein, Hsc20. This assessment favors placement of the substrate binding domain near D152 on the ATPase domain surface adjacent to the J-domain invariant HPD segment, with the Hsc70 interdomain linker in the lower cleft. Examining Hsc70 interdomain energetics, we propose that long-range electrostatic interactions, perhaps due to a difference in the pKa values of bound ATP and ADP, could play a major role in the structural change induced by ATP hydrolysis. Interdomain electrostatic interactions also appear to play a role in stimulation of ATPase activity due to J-domain binding and substrate binding by Hsc70. PMID:15273304

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain*

PubMed Central

Yates, Luke A.; Lumb, Craig N.; Brahme, Nina N.; Zalyte, Ruta; Bird, Louise E.; De Colibus, Luigi; Owens, Raymond J.; Calderwood, David A.; Sansom, Mark S. P.; Gilbert, Robert J. C.

2012-01-01

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P3; KD ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species. PMID:23132860

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

PubMed

Kandeel, Mahmoud; Kitade, Yukio

2018-02-01

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

PubMed

Vance, Tyler D R; Graham, Laurie A; Davies, Peter L

2018-04-01

Out of the dozen different ice-binding protein (IBP) structures known, the DUF3494 domain is the most widespread, having been passed many times between prokaryotic and eukaryotic microorganisms by horizontal gene transfer. This ~25-kDa β-solenoid domain with an adjacent parallel α-helix is most commonly associated with an N-terminal secretory signal peptide. However, examples of the DUF3494 domain preceded by tandem Bacterial Immunoglobulin-like (BIg) domains are sometimes found, though uncharacterized. Here, we present one such protein (SfIBP_1) from the Antarctic bacterium Shewanella frigidimarina. We have confirmed and characterized the ice-binding activity of its ice-binding domain using thermal hysteresis measurements, fluorescent ice plane affinity analysis, and ice recrystallization inhibition assays. X-ray crystallography was used to solve the structure of the SfIBP_1 ice-binding domain, to further characterize its ice-binding surface and unique method of stabilizing or 'capping' the ends of the solenoid structure. The latter is formed from the interaction of two loops mediated by a combination of tandem prolines and electrostatic interactions. Furthermore, given their domain architecture and membrane association, we propose that these BIg-containing DUF3494 IBPs serve as ice-binding adhesion proteins that are capable of adsorbing their host bacterium onto ice. Submitted new structure to the Protein Data Bank (PDB: 6BG8). © 2018 Federation of European Biochemical Societies.

Structure and DNA-Binding Sites of the SWI1 AT-rich Interaction Domain (ARID) Suggest Determinants for Sequence-Specific DNA Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Suhkmann; Zhang, Ziming; Upchurch, Sean

2004-04-16

2 ARID is a homologous family of DNA-binding domains that occur in DNA binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals and plants. SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif. The ARID domain of human SWI1 (also known as p270) does not select for a specific DNA sequence from a random sequence pool. The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences. We havemore » solved the three-dimensional structure of human SWI1 ARID using solution NMR methods. In addition, we have characterized non-specific DNA-binding by the SWI1 ARID domain. Results from this study indicate that a flexible long internal loop in ARID motif is likely to be important for sequence specific DNA-recognition. The structure of human SWI1 ARID domain also represents a distinct structural subfamily. Studies of ARID indicate that boundary of the DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins. Structural studies of homologous domains such as ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Key Words: ARID, SWI1, NMR, structural genomics, protein-DNA interaction.« less

PDZ binding to the BAR domain of PICK1 is elucidated by coarse-grained molecular dynamics.

PubMed

He, Yi; Liwo, Adam; Weinstein, Harel; Scheraga, Harold A

2011-01-07

A key regulator of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor traffic, PICK1 is known to interact with over 40 other proteins, including receptors, transporters and ionic channels, and to be active mostly as a homodimer. The current lack of a complete PICK1 structure determined at atomic resolution hinders the elucidation of its functional mechanisms. Here, we identify interactions between the component PDZ and BAR domains of PICK1 by calculating possible binding sites for the PDZ domain of PICK1 (PICK1-PDZ) to the homology-modeled, crescent-shaped dimer of the PICK1-BAR domain using multiplexed replica-exchange molecular dynamics (MREMD) and canonical molecular dynamics simulations with the coarse-grained UNRES force field. The MREMD results show that the preferred binding site for the single PDZ domain is the concave cavity of the BAR dimer. A second possible binding site is near the N-terminus of the BAR domain that is linked directly to the PDZ domain. Subsequent short canonical molecular dynamics simulations used to determine how the PICK1-PDZ domain moves to the preferred binding site on the BAR domain of PICK1 revealed that initial hydrophobic interactions drive the progress of the simulated binding. Thus, the concave face of the BAR dimer accommodates the PDZ domain first by weak hydrophobic interactions and then the PDZ domain slides to the center of the concave face, where more favorable hydrophobic interactions take over. Copyright © 2010 Elsevier Ltd. All rights reserved.

Insulator function and topological domain border strength scale with architectural protein occupancy

PubMed Central

2014-01-01

Background Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. Results By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. Conclusions We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains. PMID:24981874