Structural analysis of enzymes used for bioindustry and bioremediation.

PubMed

Tanokura, Masaru; Miyakawa, Takuya; Guan, Lijun; Hou, Feng

2015-01-01

Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

Gene expression patterns of wood decay fungi Postia placenta and Phanerochaete chrysosporium are influenced by wood substrate composition during degradation

Treesearch

Oleksandr Skyba; Daniel Cullen; Carl J. Douglas; Shawn D. Mansfield

2016-01-01

Identification of the specific genes and enzymes involved in the fungal degradation of lignocellulosic biomass derived from feedstocks with various compositions is essential to the development of improved bioenergy processes. In order to elucidate the effect of substrate composition on gene expression in wood-rotting fungi, we employed microarrays based on the...

Improved specific energy Ni-H2 cell

NASA Astrophysics Data System (ADS)

Miller, L. E.

1985-12-01

Significant improvements in specific energy for Ni-H2 battery cells have been and will be achieved. Current flight cell designs in operation on multiple satellites have achieved a specific energy of 52 Whr/Kg (this value may be compared to 45 Whr/Kg for advanced, light-weight Ni-Cd space cells). Battery cells operating at increased pressures (61 atm/900 psi) have been manufactured and successfully tested demonstrating a specific energy of 70 Whr/Kg. Further optimization of electrode substrate, pressure vessel wall thickness and cell terminal/conductor assembly designs will permit achievement of specific energies between 75-80 Whr/Kg. Energy density (outline volume) will be improved from 49 Whr/L to 79 Whr/L.

A novel SOI LDMOS with substrate field plate and variable-k dielectric buried layer

NASA Astrophysics Data System (ADS)

Li, Qi; Wen, Yi; Zhang, Fabi; Li, Haiou; Xiao, Gongli; Chen, Yonghe; Fu, Tao

2018-09-01

A novel silicon-on-insulator (SOI) lateral double-diffused metal-oxide-semiconductor (LDMOS) structure has been proposed. The new structure features a substrate field plate (SFP) and a variable-k dielectric buried layer (VKBL). The SFP and VKBL improve the breakdown voltage by introducing new electric field peaks in the surface electric field distribution. Moreover, the SFP reduces the specific ON-resistance through an enhanced auxiliary depletion effect on the drift region. The simulation results indicate that compared to the conventional SOI LDMOS structure, the breakdown voltage is improved from 118 V to 221 V, the specific ON-resistance is decreased from 7.15 mΩ·cm2 to 3.81 mΩ·cm2, the peak value of surface temperature is declined by 38 K.

Engineering Neprilysin Activity and Specificity to Create a Novel Therapeutic for Alzheimer’s Disease

PubMed Central

Webster, Carl I.; Burrell, Matthew; Olsson, Lise-Lotte; Fowler, Susan B.; Digby, Sarah; Sandercock, Alan; Snijder, Arjan; Tebbe, Jan; Haupts, Ulrich; Grudzinska, Joanna; Jermutus, Lutz; Andersson, Christin

2014-01-01

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer’s disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer’s disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1–40, 1–42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1–40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer’s disease. PMID:25089527

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

PubMed Central

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu

2013-01-01

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

QM/MM Free Energy Simulations of Salicylic Acid Methyltransferase: Effects of Stabilization of TS-like Structures on Substrate Specificity

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

Yao, Jianzhuang; Xu, Qin; Chen, Feng

2010-01-01

Salicylic acid methyltransferases (SAMTs) synthesize methyl salicylate (MeSA) using salicylate as the substrate. MeSA synthesized in plants may function as an airborne signal to activate the expression of defense-related genes and could also be a critical mobile signaling molecule that travels from the site of plant infection to establish systemic immunity in the induction of disease resistance. Here the results of QM/MM free energy simulations for the methyl transfer process in Clarkia breweri SAMT (CbSAMT) are reported to determine the origin of the substrate specificity of SAMTs. The free energy barrier for the methyl transfer from S-adenosyl-l-methionine (AdoMet) to 4-hydroxybenzoatemore » in CbSAMT is found to be about 5 kcal/mol higher than that from AdoMet to salicylate, consistent with the experimental observations. It is suggested that the relatively high efficiency for the methylation of salicylate compared to 4-hydroxybenzoate is due, at least in part, to the reason that a part of the stabilization of the transition state (TS) configuration is already reflected in the reactant complex, presumably, through the binding. The results seem to indicate that the creation of the substrate complex (e.g., through mutagenesis and substrate modifications) with its structure closely resembling TS might be fruitful for improving the catalytic efficiency for some enzymes. The results show that the computer simulations may provide important insights into the origin of the substrate specificity for the SABATH family and could be used to help experimental efforts in generating engineered enzymes with altered substrate specificity.« less

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Improved microautoradiographic method to determine individual microorganisms active in substrate uptake in natural waters.

PubMed

Tabor, P S; Neihof, R A

1982-10-01

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.

Improved Microautoradiographic Method to Determine Individual Microorganisms Active in Substrate Uptake in Natural Waters

PubMed Central

Tabor, Paul S.; Neihof, Rex A.

1982-01-01

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with 3H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (3H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method. Images PMID:16346120

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

PubMed Central

Trollope, K. M.; Görgens, J. F.

2015-01-01

The Aspergillus japonicus β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition. PMID:26253664

Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis▿†

PubMed Central

Dudek, Hanna M.; de Gonzalo, Gonzalo; Torres Pazmiño, Daniel E.; Stępniak, Piotr; Wyrwicz, Lucjan S.; Rychlewski, Leszek; Fraaije, Marco W.

2011-01-01

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope. PMID:21724896

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

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

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

PubMed

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

PubMed

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Systems and methods for using a boehmite bond-coat with polyimide membranes for gas separation

DOEpatents

Polishchuk, Kimberly Ann

2013-03-05

The subject matter disclosed herein relates to gas separation membranes and, more specifically, to polyimide gas separation membranes. In an embodiment, a gas separation membrane includes a porous substrate, a substantially continuous polyimide membrane layer, and one or more layers of boehmite nanoparticles disposed between the porous substrate and the polyimide membrane layer to form a bond-coat layer. The bond-coat layer is configured to improve the adhesion of the polyimide membrane layer to the porous substrate, and the polyimide membrane layer has a thickness approximately 100 nm or less.

High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.

PubMed

Xing, Yang; Bu, Lingxi; Sun, Dafeng; Liu, Zhiping; Liu, Shijie; Jiang, Jianxin

2015-10-01

This study reports four schemes to pretreat wet furfural residues (FRs) with sodium bisulfite for production of fermentable sugar. The results showed that non-detoxified FRs (pH 2-3) had great potential to lower the cost of bioconversion. The optimal process was that unwashed FRs were first pretreated with bisulfite, and the whole slurry was then directly used for enzymatic hydrolysis. A maximum glucose yield of 99.4% was achieved from substrates pretreated with 0.1 g NaHSO3/g dry substrate (DS), at a relatively low temperature of 100 °C for 3 h. Compared with raw material, enzymatic hydrolysis at a high-solid of 16.5% (w/w) specifically showed more excellent performance with bisulfite treated FRs. Direct bisulfite pretreatment improved the accessibility of substrates and the total glucose recovery. Lignosulfonate in the non-detoxified slurry decreased the non-productive adsorption of cellulase on the substrate, thus improving enzymatic hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

X-Ray Computed Tomography Reveals the Response of Root System Architecture to Soil Texture1[OPEN

PubMed Central

Rogers, Eric D.; Monaenkova, Daria; Mijar, Medhavinee; Goldman, Daniel I.

2016-01-01

Root system architecture (RSA) impacts plant fitness and crop yield by facilitating efficient nutrient and water uptake from the soil. A better understanding of the effects of soil on RSA could improve crop productivity by matching roots to their soil environment. We used x-ray computed tomography to perform a detailed three-dimensional quantification of changes in rice (Oryza sativa) RSA in response to the physical properties of a granular substrate. We characterized the RSA of eight rice cultivars in five different growth substrates and determined that RSA is the result of interactions between genotype and growth environment. We identified cultivar-specific changes in RSA in response to changing growth substrate texture. The cultivar Azucena exhibited low RSA plasticity in all growth substrates, whereas cultivar Bala root depth was a function of soil hardness. Our imaging techniques provide a framework to study RSA in different growth environments, the results of which can be used to improve root traits with agronomic potential. PMID:27208237

Progress toward thin-film GaAs solar cells using a single-crystal Si substrate with a Ge interlayer

NASA Technical Reports Server (NTRS)

Yeh, Y. C. M.; Wang, K. L.; Zwerdling, S.

1982-01-01

Development of a technology for fabricating light-weight, high-efficiency, radiation-resistant solar cells for space applications is reported. The approaches currently adopted are to fabricate shallow homojunction n(+)/p as well as p/n AlGaAs-heteroface GaAs solar cells by organometallic chemical vapor deposition (OM-CVD) on single-crystal Si substrates using in each case, a thin Ge epi-interlayer first grown by CVD. This approach maintains the advantages of the low specific gravity of Si as well as the high efficiency and radiation-resistant properties of the GaAs solar cell which can lead to greatly improved specific power for a solar array. The growth of single-crystal GaAs epilayers on Ge epi-interlayers on Si substrates is investigated. Related solar cell fabrication is reviewed.

Identification of New Drug Targets in Multi-Drug Resistant Bacterial Infections

DTIC Science & Technology

2013-10-01

observation allowed us to improve the substrate specificity assay and identify that threonine serves as an optimal substrate in the pyrophosphate exchange...activity was obtained, and the activity now seems to be optimal with the small hydroxyl containing amino acids threonine and serine. These... diabetic ulcers are favored sites of infection (6, 7). A. baumannii has also been shown to cause infections outside the health care setting, namely, severe

Conceptual design study of concentrator enhanced solar arrays for space applications. 2kW Si and GaAs systems at 1 AU

NASA Technical Reports Server (NTRS)

1980-01-01

The effect of concentration level on the specific power for a deployable, thin, gallium arsenide cell array in geosynchronous orbit for 10 years in conjunction with a two dimensional flat plate trough concentrator (V trough) and also with a multiple flat plate concentrator was investigated as well as the effects for a conventional silicon cell array on a rigid substrate. For application to a thin GaAs array at 1 AU for 10 years, the V trough produces a 19% benefit in specific power and a dramatic reduction in array area, while the multiple flat plate collector design is not only of no benefit, but is a considerable detriment. The benefit it achieves by reducing array area is duplicated by the 2D design. For the silicon array on a rigid substrate, improvement in performance due to a concentrator with ordinary mirror coating is quite small: 9% increase in specific power, and 13% reduction in array area. When the concentrator mirrors are coated with an improved cold mirror coating, somewhat more significant results are obtained: 31% specific power improvement; and 27% area reduction. In both cases, a 10 year exposure reduces BOL output by 23%.

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

PubMed Central

Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

2015-01-01

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy. PMID:25793518

Workshop on Heteroepitaxial InP Solar Cells

NASA Technical Reports Server (NTRS)

Weinberg, I.; Walters, R. W.

1993-01-01

In a generic sense, the justification for any sort of InP solar cell research applies, i.e. to take advantage of the inherently high radiation resistance and efficiency of InP solar cells. To be more specific, the approach is justified by its potential for significant cost reduction and the availability of greatly increased cell area afforded by substrates such as Si and Ge. The use of substrates, such as the latter two, would result in increased ruggedness, ease of handling, and improved manufacturability. The use of more rugged substrates would lead to a greatly increased capability for cell thinning leading to the desirable feature of reduced array weight.

Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

PubMed

Chen, Ke-Cai; Zheng, Ming-Min; Pan, Jiang; Li, Chun-Xiu; Xu, Jian-He

2017-10-01

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipA L315S and LipA S271F , were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipA L315S , LipA S271F , and their combination LipA L315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipA S271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipA S271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m . Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

Nickel foam-supported polyaniline cathode prepared with electrophoresis for improvement of rechargeable Zn battery performance

NASA Astrophysics Data System (ADS)

Xia, Yang; Zhu, Derong; Si, Shihui; Li, Degeng; Wu, Sen

2015-06-01

Porous nickel foam is used as a substrate for the development of rechargeable zinc//polyaniline battery, and the cathode electrophoresis of PANI microparticles in non-aqueous solution is applied to the fabrication of Ni foam supported PANI electrode, in which the corrosion of the nickel foam substrate is prohibited. The Ni foam supported PANI cathode with high loading is prepared by PANI electrophoretic deposition, and followed by PANI slurry casting under vacuum filtration. The electrochemical charge storage performance for PANI material is significantly improved by using nickel foam substrate via the electrophoretic interlayer. The specific capacity of the nickel foam-PANI electrode with the electrophoretic layer is higher than the composite electrode without the electrophoretic layer, and the specific capacity of PANI supported by Ni foam reaches up to 183.28 mAh g-1 at the working current of 2.5 mA cm-2. The present electrophoresis deposition method plays the facile procedure for the immobilization of PANI microparticles onto the surface of non-platinum metals, and it becomes feasible to the use of the Ni foam supported PANI composite cathode for the Zn/PANI battery in weak acidic electrolyte.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

PubMed Central

Jansen, Mickel L. A.; Daran-Lapujade, Pascale; de Winde, Johannes H.; Piper, Matthew D. W.; Pronk, Jack T.

2004-01-01

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. PMID:15066785

Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

PubMed Central

2015-01-01

The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield. PMID:24804152

Substrate stiffness affects skeletal myoblast differentiation in vitro

NASA Astrophysics Data System (ADS)

Romanazzo, Sara; Forte, Giancarlo; Ebara, Mitsuhiro; Uto, Koichiro; Pagliari, Stefania; Aoyagi, Takao; Traversa, Enrico; Taniguchi, Akiyoshi

2012-12-01

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ɛ-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

Improvement of antigen detection efficiency with the use of two-dimensional photonic crystal as a substrate

NASA Astrophysics Data System (ADS)

Dovzhenko, Dmitriy; Terekhin, Vladimir; Vokhmincev, Kirill; Sukhanova, Alyona; Nabiev, Igor

2017-01-01

Multiplex detection of different antigens in human serum in order to reveal diseases at the early stage is of interest nowadays. There are a lot of biosensors, which use the fluorescent labels for specific detection of analytes. For instance, common method for detection of antigens in human serum samples is enzyme-linked immunosorbent assay (ELISA). One of the most effective ways to improve the sensitivity of this detection method is the use of a substrate that could enhance the fluorescent signal and make it easier to collect. Two-dimensional (2D) photonic crystals are very suitable structures for these purposes because of the ability to enhance the luminescent signal, control the light propagation and perform the analysis directly on its surface. In our study we have calculated optimal parameters for 2D-dimensional photonic crystal consisting of the array of silicon nano-rods, fabricated such photonic crystal on a silicon substrate using reactive ion etching and showed the possibility of its efficient application as a substrate for ELISA detection of human cancer antigens.

X-Ray Computed Tomography Reveals the Response of Root System Architecture to Soil Texture.

PubMed

Rogers, Eric D; Monaenkova, Daria; Mijar, Medhavinee; Nori, Apoorva; Goldman, Daniel I; Benfey, Philip N

2016-07-01

Root system architecture (RSA) impacts plant fitness and crop yield by facilitating efficient nutrient and water uptake from the soil. A better understanding of the effects of soil on RSA could improve crop productivity by matching roots to their soil environment. We used x-ray computed tomography to perform a detailed three-dimensional quantification of changes in rice (Oryza sativa) RSA in response to the physical properties of a granular substrate. We characterized the RSA of eight rice cultivars in five different growth substrates and determined that RSA is the result of interactions between genotype and growth environment. We identified cultivar-specific changes in RSA in response to changing growth substrate texture. The cultivar Azucena exhibited low RSA plasticity in all growth substrates, whereas cultivar Bala root depth was a function of soil hardness. Our imaging techniques provide a framework to study RSA in different growth environments, the results of which can be used to improve root traits with agronomic potential. © 2016 American Society of Plant Biologists. All Rights Reserved.

Toward an Increased Functionality in Oyster ( Pleurotus) Mushrooms Produced on Grape Marc or Olive Mill Wastes Serving as Sources of Bioactive Compounds.

PubMed

Koutrotsios, Georgios; Kalogeropoulos, Nick; Kaliora, Andriana C; Zervakis, Georgios I

2018-06-20

Pleurotus ostreatus, P. eryngii, and P. nebrodensis were cultivated on nonconventional substrates containing grape marc (GMC) or olive mill byproducts (OMB); wheat straw (WHS) served as control. GMC-based media demonstrated equal/better mushroom productivity than WHS for P. eryngii and P. nebrodensis, while the cultivation performance of P. eryngii was improved in OMB-based media. Both GMC and OMB substrates led to large increase of fruit-bodies content in phenolic acids, resveratrol, triterpenic compounds, and ergosterol; in particular, P. eryngii mushrooms presented significantly more total phenolics and exhibited much higher antioxidant activity (2- to 8-fold increase). Furthermore, substrates containing GMC or OMB presented up to 27% increase in mushroom β-glucans. Overall, Pleurotus species responded in a different and mostly substrate-specific manner by selectively absorbing organic compounds. Phenolics and squalene content of substrates correlated very well with mushrooms antioxidant activity and ergosterol, respectively; the same was observed for triterpenics' content of substrates and mushrooms.

A systemic study on key parameters affecting nanocomposite coatings on magnesium substrates.

PubMed

Johnson, Ian; Wang, Sebo Michelle; Silken, Christine; Liu, Huinan

2016-05-01

Nanocomposite coatings offer multiple functions simultaneously to improve the interfacial properties of magnesium (Mg) alloys for skeletal implant applications, e.g., controlling the degradation rate of Mg substrates, improving bone cell functions, and providing drug delivery capability. However, the effective service time of nanocomposite coatings may be limited due to their early delamination from the Mg-based substrates. Therefore, the objective of this study was to address the delamination issue of nanocomposite coatings, improve the coating properties for reducing the degradation of Mg-based substrates, and thus improve their cytocompatibility with bone marrow derived mesenchymal stem cells (BMSCs). The surface conditions of the substrates, polymer component type of the nanocomposite coatings, and post-deposition processing are the key parameters that contribute to the efficacy of the nanocomposite coatings in regulating substrate degradation and bone cell responses. Specifically, the effects of metallic surface versus alkaline heat-treated hydroxide surface of the substrates on coating quality were investigated. For the nanocomposite coatings, nanophase hydroxyapatite (nHA) was dispersed in three types of biodegradable polymers, i.e., poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid) (PLLA), or poly(caprolactone) (PCL) to determine which polymer component could provide integrated properties for slowest Mg degradation. The nanocomposite coatings with or without post-deposition processing, i.e., melting, annealing, were compared to determine which processing route improved the properties of the nanocomposite coatings most significantly. The results showed that optimizing the coating processes addressed the delamination issue. The melted then annealed nHA/PCL coating on the metallic Mg substrates showed the slowest degradation and the best coating adhesion, among all the combinations of conditions studied; and, it improved the adhesion density of BMSCs. This study elucidated the key parameters for optimizing nanocomposite coatings on Mg-based substrates for skeletal implant applications, and provided rational design guidelines for the nanocomposite coatings on Mg alloys for potential clinical translation of biodegradable Mg-based implants. This manuscript describes the systemic optimization of nanocomposite coatings to control the degradation and bioactivity of magnesium for skeletal implant applications. The key parameters influencing the integrity and functions of the nanocomposite coatings on magnesium were identified, guidelines for the optimization of the coatings were established, and the benefits of coating optimization were demonstrated through reduced magnesium degradation and increased bone marrow derived mesenchymal stem cell (BMSC) adhesion in vitro. The guidelines developed in this manuscript are valuable for the biometal field to improve the design of bioresorbable implants and devices, which will advance the clinical translation of magnesium-based implants. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

Influence of the size reduction of organic waste on their anaerobic digestion.

PubMed

Palmowski, L M; Müller, J A

2000-01-01

The rate-limiting step in anaerobic digestion of organic solid waste is generally their hydrolysis. A size reduction of the particles and the resulting enlargement of the available specific surface can support the biological process in two ways. Firstly, in case of substrates with a high content of fibres and a low xegradability, their comminution yields to an improved digester gas production. This leads to a decreased amount of residues to be disposed of and to an increased quantity of useful digester gas. The second effect of the particle size reduction observed with all the substrates but particularly with those of low degradability is a reduction of the technical digestion time. Furthermore, the particle size of organic waste has an influence on the dewaterability after codigestion with sewage sludge. The presence of organic waste residues improves the dewaterability measured as specific resistance to filtration but this positive effect is attenuated if the particle size of the solids is reduced.

Substrate Specifity Profiling of the Aspergillus fumigatus Proteolytic Secretome Reveals Consensus Motifs with Predominance of Ile/Leu and Phe/Tyr

PubMed Central

Watson, Douglas S.; Feng, Xizhi; Askew, David S.; Jambunathan, Kalyani; Kodukula, Krishna; Galande, Amit K.

2011-01-01

Background The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers. Methodology and Principal Findings As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures. Conclusions This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis. PMID:21695046

Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence.

PubMed

Masuyer, Geoffrey; Yates, Christopher J; Sturrock, Edward D; Acharya, K Ravi

2014-10-01

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

Substrate specificity and thermostability of the dehairing alkaline protease from Bacillus pumilus.

PubMed

Wan, Min-Yuan; Wang, Hai-Yan; Zhang, Yi-Zheng; Feng, Hong

2009-11-01

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k(m) value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 degrees C, and lost activity so quickly above 70 degrees C. The thermostability was largely dependent on metal ion, especially Ca(2+), although other ions, like Mg(2+), Mn(2+), and Co(2+), could sustain stability at certain extent within limited time. Cu(2+), Fe(2+), as well as Al(3+), did not support the enzyme to retain activity at 60 degrees C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

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

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

A multiphase mixture model for substrate concentration distribution characteristics and photo-hydrogen production performance of the entrapped-cell photobioreactor.

PubMed

Guo, Cheng-Long; Cao, Hong-Xia; Pei, Hong-Shan; Guo, Fei-Qiang; Liu, Da-Meng

2015-04-01

A multiphase mixture model was developed for revealing the interaction mechanism between biochemical reactions and transfer processes in the entrapped-cell photobioreactor packed with gel granules containing Rhodopseudomonas palustris CQK 01. The effects of difference operation parameters, including operation temperature, influent medium pH value and porosity of packed bed, on substrate concentration distribution characteristics and photo-hydrogen production performance were investigated. The results showed that the model predictions were in good agreement with the experimental data reported. Moreover, the operation temperature of 30 °C and the influent medium pH value of 7 were the most suitable conditions for photo-hydrogen production by biodegrading substrate. In addition, the lower porosity of packed bed was beneficial to enhance photo-hydrogen production performance owing to the improvement on the amount of substrate transferred into gel granules caused by the increased specific area for substrate transfer in the elemental volume. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bovine Milk as a Source of Functional Oligosaccharides for Improving Human Health12

PubMed Central

Zivkovic, Angela M.; Barile, Daniela

2011-01-01

Human milk oligosaccharides are complex sugars that function as selective growth substrates for specific beneficial bacteria in the gastrointestinal system. Bovine milk is a potentially excellent source of commercially viable analogs of these unique molecules. However, bovine milk has a much lower concentration of these oligosaccharides than human milk, and the majority of the molecules are simpler in structure than those found in human milk. Specific structural characteristics of milk-derived oligosaccharides are crucial to their ability to selectively enrich beneficial bacteria while inhibiting or being less than ideal substrates for undesirable and pathogenic bacteria. Thus, if bovine milk products are to provide human milk–like benefits, it is important to identify specific dairy streams that can be processed commercially and cost-effectively and that can yield specific oligosaccharide compositions that will be beneficial as new food ingredients or supplements to improve human health. Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk. With further refinements to dairy stream processing techniques and functional testing to identify streams that are particularly suitable for enriching beneficial intestinal bacteria, the future of oligosaccharides isolated from dairy streams as a food category with substantiated health claims is promising. PMID:22332060

Bovine milk as a source of functional oligosaccharides for improving human health.

PubMed

Zivkovic, Angela M; Barile, Daniela

2011-05-01

Human milk oligosaccharides are complex sugars that function as selective growth substrates for specific beneficial bacteria in the gastrointestinal system. Bovine milk is a potentially excellent source of commercially viable analogs of these unique molecules. However, bovine milk has a much lower concentration of these oligosaccharides than human milk, and the majority of the molecules are simpler in structure than those found in human milk. Specific structural characteristics of milk-derived oligosaccharides are crucial to their ability to selectively enrich beneficial bacteria while inhibiting or being less than ideal substrates for undesirable and pathogenic bacteria. Thus, if bovine milk products are to provide human milk-like benefits, it is important to identify specific dairy streams that can be processed commercially and cost-effectively and that can yield specific oligosaccharide compositions that will be beneficial as new food ingredients or supplements to improve human health. Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk. With further refinements to dairy stream processing techniques and functional testing to identify streams that are particularly suitable for enriching beneficial intestinal bacteria, the future of oligosaccharides isolated from dairy streams as a food category with substantiated health claims is promising.

Evaluating the Atrial Myopathy Underlying Atrial Fibrillation: Identifying the Arrhythmogenic and Thrombogenic Substrate

PubMed Central

Goldberger, Jeffrey J.; Arora, Rishi; Green, David; Greenland, Philip; Lee, Daniel C.; Lloyd-Jones, Donald M.; Markl, Michael; Ng, Jason; Shah, Sanjiv J.

2015-01-01

Atrial disease or myopathy forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. Current diagnostic approaches in patients with AF focus on identifying clinical predictors with evaluation of left atrial size by echocardiography serving as the sole measure specifically evaluating the atrium. Although the atrial substrate underlying AF is likely developing for years prior to the onset of AF, there is no current evaluation to identify the pre-clinical atrial myopathy. Atrial fibrosis is one component of the atrial substrate that has garnered recent attention based on newer MRI techniques that have been applied to visualize atrial fibrosis in humans with prognostic implications regarding success of treatment. Advanced ECG signal processing, echocardiographic techniques, and MRI imaging of fibrosis and flow provide up-to-date approaches to evaluate the atrial myopathy underlying AF. While thromboembolic risk is currently defined by clinical scores, their predictive value is mediocre. Evaluation of stasis via imaging and biomarkers associated with thrombogenesis may provide enhanced approaches to assess risk for stroke in patients with AF. Better delineation of the atrial myopathy that serves as the substrate for AF and thromboembolic complications might improve treatment outcomes. Furthermore, better delineation of the pathophysiologic mechanisms underlying the development of the atrial substrate for AF, particularly in its earlier stages, could help identify blood and imaging biomarkers that could be useful to assess risk for developing new onset AF and suggest specific pathways that could be targeted for prevention. PMID:26216085

Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates

PubMed Central

Huang, Ruiqi; Hippauf, Frank; Rohrbeck, Diana; Haustein, Maria; Wenke, Katrin; Feike, Janie; Sorrelle, Noah; Piechulla, Birgit; Barkman, Todd J.

2012-01-01

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (kcat/KM) for competing substrates, even though adaptive substitutions may affect KM and kcat separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities. PMID:22315396

Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates.

PubMed

Huang, Ruiqi; Hippauf, Frank; Rohrbeck, Diana; Haustein, Maria; Wenke, Katrin; Feike, Janie; Sorrelle, Noah; Piechulla, Birgit; Barkman, Todd J

2012-02-21

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (k(cat)/K(M)) for competing substrates, even though adaptive substitutions may affect K(M) and k(cat) separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities.

Batch anaerobic co-digestion of waste activated sludge and microalgae (Chlorella sorokiniana) at mesophilic temperature.

PubMed

Beltrán, Carolina; Jeison, David; Fermoso, Fernando G; Borja, Rafael

2016-08-23

The microalgae Chlorella sorokiniana are used as co-substrate for waste activated sludge (WAS) anaerobic digestion. The specific objective of this research was to evaluate the feasibility of improving methane production from anaerobic digestion of WAS in co-digestion with this microalga, based on an optimized mixture percentage. Thus, the anaerobic co-digestion of both substrates aims to overcome the drawbacks of the anaerobic digestion of single WAS, simultaneously improving its management. Different co-digestion mixtures (0% WAS-100% microalgae; 25% WAS-75% microalgae; 50% WAS-50% microalgae; 75% WAS-25% microalgae; 100% WAS-0% microalgae) were studied. The highest methane yield (442 mL CH4/g VS) was obtained for the mixture with 75% WAS and 25% microalgae. This value was 22% and 39% higher than that obtained in the anaerobic digestion of the sole substrates WAS and microalgae, respectively, as well as 16% and 25% higher than those obtained for the co-digestion mixtures with 25% WAS and 75% microalgae and 50% WAS and 50% microalgae, respectively. The kinetic constant of the process increased 42%, 42% and 12%, respectively, for the mixtures with 25%, 50% and 75% of WAS compared to the substrate without WAS. Anaerobic digestion of WAS, together with C. sorokiniana, has been clearly improved by ensuring its viability, suitability and efficiency.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, Frances H.; Moore, Jeffrey C.

1998-01-01

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, Frances H.; Moore, Jeffrey C.

1999-01-01

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

Both Intrinsic Substrate Preference and Network Context Contribute to Substrate Selection of Classical Tyrosine Phosphatases*

PubMed Central

Tinti, Michele; Paoluzi, Serena; Santonico, Elena; Masch, Antonia; Schutkowski, Mike

2017-01-01

Reversible tyrosine phosphorylation is a widespread post-translational modification mechanism underlying cell physiology. Thus, understanding the mechanisms responsible for substrate selection by kinases and phosphatases is central to our ability to model signal transduction at a system level. Classical protein-tyrosine phosphatases can exhibit substrate specificity in vivo by combining intrinsic enzymatic specificity with the network of protein-protein interactions, which positions the enzymes in close proximity to their substrates. Here we use a high throughput approach, based on high density phosphopeptide chips, to determine the in vitro substrate preference of 16 members of the protein-tyrosine phosphatase family. This approach helped identify one residue in the substrate binding pocket of the phosphatase domain that confers specificity for phosphopeptides in a specific sequence context. We also present a Bayesian model that combines intrinsic enzymatic specificity and interaction information in the context of the human protein interaction network to infer new phosphatase substrates at the proteome level. PMID:28159843

Solid state fermentation and production of rifamycin SV using Amycolatopsis mediterranei.

PubMed

Nagavalli, M; Ponamgi, S P D; Girijashankar, V; Venkateswar Rao, L

2015-01-01

Production of Rifamycin SV from cheaper agro-industrial by-products using mutant strain of Amycolatopsis mediterranei OVA5-E7 in solid state fermentation (SSF) was optimized. Among the agro-based substrates used, ragi bran was found suitable for maximizing the yield of Rifamycin SV (1310 mg 100 g(-1) ds). The yield can be further enhanced to 19·7 g Kg(-1) of dry substrate by supplementing the substrate with deoiled cotton cake (10% w/w) using optimized fermentation parameters such as maintaining 80% moisture, pH 7·0, 30°C incubation temperature, inoculum 25% v/w and carrying the solid state fermenting for 9 days. Manipulating these seven specifications, the end product yield achieved in our experimentation was 20 g of Rifamycin SV Kg(-1) ds. Eventually, an overall 5-fold improvement in Rifamycin SV production was achieved. Antibiotics such as rifamycin are broad-spectrum antimicrobial drugs used in large-scale worldwide as human medicine towards controlling diseases. Amycolatopsis mediterranei strain which produces this antibiotic was earlier used in submerged fermentation yielded lower amounts of rifamycin. By employing cheaper agro-industrial by-products, we produced upto 20 g rifamycin SV per Kg dry substrate used under optimized solid state fermentation conditions. Keeping in view, the role of rifamycin in meeting the medical demands of world's increasing population; we successfully used an improved strain on cheaper substrates with optimized fermentation parameters and achieved a 5-fold improvement in rifamycin SV production. © 2014 The Society for Applied Microbiology.

Directed evolution to re-adapt a co-evolved network within an enzyme.

PubMed

Strafford, John; Payongsri, Panwajee; Hibbert, Edward G; Morris, Phattaraporn; Batth, Sukhjeet S; Steadman, David; Smith, Mark E B; Ward, John M; Hailes, Helen C; Dalby, Paul A

2012-01-01

We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k(cat) 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network. Copyright © 2011 Elsevier B.V. All rights reserved.

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

PubMed

Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

2016-09-13

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

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

Boernke, W. E.; Millard, C. S.; Stevens, P. W.

1995-09-10

Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the nativemore » enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant malate dehydrogenase. However, when expressed in a strain of E. coli unable to ferment glucose, the mutant enzyme restored growth and produced lactic acid as the sole fermentation product.« less

Targeted mutations and MD simulations of a methanol-stable lipase YLIP9 from Yarrowia lipolytica MSR80 to develop a biodiesel enzyme.

PubMed

Syal, Poonam; Verma, Ved Vrat; Gupta, Rani

2017-11-01

Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity. In this mutant, six binding pocket residues (Bp2-Bp7) were further mutated to obtain six double mutants. YLIP9L1Bp3 showed significant shift in substrate specificity towards long-chain pNPesters with 11-fold increase in catalytic efficiency than YLIP9. Double mutations also led to increased thermostability and lowered activation energy of YLIP9L1Bp3 thereby shifting its optimum temperature from 60°C to 50°C. In silico molecular dynamics simulations revealed improved lid flexibility and increased catalytic triad volume in YLIP9L1Bp3. The enzyme YLIP9L1Bp3 was methanol-stable having selectivity for long-chain fatty acids with improved catalytic efficiency. Its application as a biodiesel enzyme was validated by transesterification of palm oil in presence of methanol, where it showed 8-fold increase in conversion of oil to methyl esters. Copyright © 2017 Elsevier B.V. All rights reserved.

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. We performed a principal component analysis using 1-nanosecond molecular dynamics simulations using solvent boundary condition. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity.

Control of Growth Rate by Initial Substrate Concentration at Values Below Maximum Rate

PubMed Central

Gaudy, Anthony F.; Obayashi, Alan; Gaudy, Elizabeth T.

1971-01-01

The hyperbolic relationship between specific growth rate, μ, and substrate concentration, proposed by Monod and used since as the basis for the theory of steady-state growth in continuous-flow systems, was tested experimentally in batch cultures. Use of a Flavobacterium sp. exhibiting a high saturation constant for growth in glucose minimal medium allowed direct measurement of growth rate and substrate concentration throughout the growth cycle in medium containing a rate-limiting initial concentration of glucose. Specific growth rates were also measured for a wide range of initial glucose concentrations. A plot of specific growth rate versus initial substrate concentration was found to fit the hyperbolic equation. However, the instantaneous relationship between specific growth rate and substrate concentration during growth, which is stated by the equation, was not observed. Well defined exponential growth phases were developed at initial substrate concentrations below that required for support of the maximum exponential growth rate and a constant doubling time was maintained until 50% of the substrate had been used. It is suggested that the external substrate concentration initially present “sets” the specific growth rate by establishing a steady-state internal concentration of substrate, possibly through control of the number of permeation sites. PMID:5137579

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.

PubMed

Hayashi, Junji; Seto, Tomonari; Akita, Hironaga; Watanabe, Masahiro; Hoshino, Tamotsu; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

2017-06-01

A stable NADP + -dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P) + -dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. Copyright © 2017 American Society for Microbiology.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase

PubMed Central

Hayashi, Junji; Seto, Tomonari; Akita, Hironaga; Watanabe, Masahiro; Hoshino, Tamotsu; Yoneda, Kazunari; Ohshima, Toshihisa

2017-01-01

ABSTRACT A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. PMID:28363957

Superhydrophobic SERS substrates based on silicon hierarchical nanostructures

NASA Astrophysics Data System (ADS)

Chen, Xuexian; Wen, Jinxiu; Zhou, Jianhua; Zheng, Zebo; An, Di; Wang, Hao; Xie, Weiguang; Zhan, Runze; Xu, Ningsheng; Chen, Jun; She, Juncong; Chen, Huanjun; Deng, Shaozhi

2018-02-01

Silicon nanostructures have been cultivated as promising surface enhanced Raman scattering (SERS) substrates in terms of their low-loss optical resonance modes, facile functionalization, and compatibility with today’s state-of-the-art CMOS techniques. However, unlike their plasmonic counterparts, the electromagnetic field enhancements induced by silicon nanostructures are relatively small, which restrict their SERS sensing limit to around 10-7 M. To tackle this problem, we propose here a strategy for improving the SERS performance of silicon nanostructures by constructing silicon hierarchical nanostructures with a superhydrophobic surface. The hierarchical nanostructures are binary structures consisted of silicon nanowires (NWs) grown on micropyramids (MPs). After being modified with perfluorooctyltriethoxysilane (PFOT), the nanostructure surface shows a stable superhydrophobicity with a high contact angle of ˜160°. The substrate can allow for concentrating diluted analyte solutions into a specific area during the evaporation of the liquid droplet, whereby the analytes are aggregated into a small volume and can be easily detected by the silicon nanostructure SERS substrate. The analyte molecules (methylene blue: MB) enriched from an aqueous solution lower than 10-8 M can be readily detected. Such a detection limit is ˜100-fold lower than the conventional SERS substrates made of silicon nanostructures. Additionally, the detection limit can be further improved by functionalizing gold nanoparticles onto silicon hierarchical nanostructures, whereby the superhydrophobic characteristics and plasmonic field enhancements can be combined synergistically to give a detection limit down to ˜10-11 M. A gold nanoparticle-functionalized superhydrophobic substrate was employed to detect the spiked melamine in liquid milk. The results showed that the detection limit can be as low as 10-5 M, highlighting the potential of the proposed superhydrophobic SERS substrate in practical food safety inspection applications.

Plasmonic crystal based solid substrate for biomedical application of SERS

NASA Astrophysics Data System (ADS)

Morasso, Carlo F.; Mehn, Dora; Picciolini, Silvia; Vanna, Renzo; Bedoni, Marzia; Gramatica, Furio; Pellacani, Paola; Frangolho, Ana; Marchesini, Gerardo; Valsesia, Andrea

2014-02-01

Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson's disease which has been demonstrated being difficult to study by traditional HPLC based approaches.

On-chip ultra-thin layer chromatography and surface enhanced Raman spectroscopy.

PubMed

Chen, Jing; Abell, Justin; Huang, Yao-wen; Zhao, Yiping

2012-09-07

We demonstrate that silver nanorod (AgNR) array substrates can be used for on-chip separation and detection of chemical mixtures by combining ultra-thin layer chromatography (UTLC) and surface enhanced Raman spectroscopy (SERS). The UTLC-SERS plate consists of an AgNR array fabricated by oblique angle deposition. The capability of the AgNR substrates to separate the different compounds in a mixture was explored using a mixture of four dyes and a mixture of melamine and Rhodamine 6G at varied concentrations with different mobile phase solvents. After UTLC separation, spatially-resolved SERS spectra were collected along the mobile phase development direction and the intensities of specific SERS peaks from each component were used to generate chromatograms. The AgNR substrates demonstrate the potential for separating the test dyes with plate heights as low as 9.6 μm. The limits of detection are between 10(-5)-10(-6) M. Furthermore, we show that the coupling of UTLC with SERS improves the SERS detection specificity, as small amounts of target analytes can be separated from the interfering background components.

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

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

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE PAGES

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.; ...

2017-02-01

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.

PubMed

Kim, Doyoun; San, Boi Hoa; Moh, Sang Hyun; Park, Hyejin; Kim, Dong Young; Lee, Sangho; Kim, Kyeong Kyu

2010-01-01

Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA. Copyright 2009 Elsevier Inc. All rights reserved.

Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

ERIC Educational Resources Information Center

Kin, Ng Hong; Ling, Tan Aik

2016-01-01

The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

Dropwise Condensation on Soft Hydrophobic Coatings.

PubMed

Phadnis, Akshay; Rykaczewski, Konrad

2017-10-31

Promoting dropwise condensation (DWC) could improve the efficiency of many industrial systems. Consequently, a lot of effort has been dedicated to finding durable materials that could sustainably promote DWC as well as finding routes to enhance the heat transfer rate during this phase change process. Motivated by previous reports of substrate softening increasing droplet nucleation rate, here we investigated how mechanical properties of a substrate impact relevant droplet-surface interactions and DWC heat transfer rate. Specifically, we experimentally quantified the effect of hydrophobic elastomer's shear modulus on droplet nucleation density and shedding radius. To quantify the impact of substrate softening on heat transfer through individual droplets, we combined analytical solution of elastomer deformation induced by droplets with finite element modeling of the heat transfer process. By substituting these experimentally and theoretically derived values into DWC heat transfer model, we quantified the compounding effect of the substrate's mechanical properties on the overall heat transfer rate. Our results show that softening of the substrates below a shear modulus of 500 kPa results in a significant reduction in the condensation heat transfer rate. This trend is primarily driven by additional thermal resistance of the liquid posed by depression of the soft substrate.

Removal of pharmaceuticals in pre-denitrifying MBBR - Influence of organic substrate availability in single- and three-stage configurations.

PubMed

Polesel, Fabio; Torresi, Elena; Loreggian, Luca; Casas, Mònica Escolà; Christensson, Magnus; Bester, Kai; Plósz, Benedek Gy

2017-10-15

Due to the limited efficiency of conventional biological treatment, innovative solutions are being explored to improve the removal of trace organic chemicals in wastewater. Controlling biomass exposure to growth substrate represents an appealing option for process optimization, as substrate availability likely impacts microbial activity, hence organic trace chemical removal. This study investigated the elimination of pharmaceuticals in pre-denitrifying moving bed biofilm reactors (MBBRs), where biofilm exposure to different organic substrate loading and composition was controlled by reactor staging. A three-stage MBBR and a single-stage reference MBBR (with the same operating volume and filling ratio) were operated under continuous-flow conditions (18 months). Two sets of batch experiments (day 100 and 471) were performed to quantify and compare pharmaceutical removal and denitrification kinetics in the different MBBRs. Experimental results revealed the possible influence of retransformation (e.g., from conjugated metabolites) and enantioselectivity on the removal of selected pharmaceuticals. In the second set of experiments, specific trends in denitrification and biotransformation kinetics were observed, with highest and lowest rates/rate constants in the first (S1) and the last (S3) staged sub-reactors, respectively. These observations were confirmed by removal efficiency data obtained during continuous-flow operation, with limited removal (<10%) of recalcitrant pharmaceuticals and highest removal in S1 within the three-stage MBBR. Notably, biotransformation rate constants obtained for non-recalcitrant pharmaceuticals correlated with mean specific denitrification rates, maximum specific growth rates and observed growth yield values. Overall, these findings suggest that: (i) the long-term exposure to tiered substrate accessibility in the three-stage configuration shaped the denitrification and biotransformation capacity of biofilms, with significant reduction under substrate limitation; (ii) biotransformation of pharmaceuticals may have occurred as a result of cometabolism by heterotrophic denitrifying bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Impact of substrate etching on plasmonic elements and metamaterials: preventing red shift and improving refractive index sensitivity.

PubMed

Moritake, Yuto; Tanaka, Takuo

2018-02-05

We propose and demonstrate the elimination of substrate influence on plasmon resonance by using selective and isotropic etching of substrates. Preventing the red shift of the resonance due to substrates and improving refractive index sensitivity were experimentally demonstrated by using plasmonic nanostructures fabricated on silicon substrates. Applying substrate etching decreases the effective refractive index around the metal nanostructures, resulting in elimination of the red shift. Improvement of sensitivity to the refractive index environment was demonstrated by using plasmonic metamaterials with Fano resonance based on far field interference. Change in quality factors (Q-factors) of the Fano resonance by substrate etching was also investigated in detail. The presence of a closely positioned substrate distorts the electric field distribution and degrades the Q-factors. Substrate etching dramatically increased the refractive index sensitivity reaching to 1532 nm/RIU since the electric fields under the nanostructures became accessible through substrate etching. The FOM was improved compared to the case without the substrate etching. The method presented in this paper is applicable to a variety of plasmonic structures to eliminate the influence of substrates for realizing high performance plasmonic devices.

Co-digestion of municipal sewage sludge and solid waste: modelling of carbohydrate, lipid and protein content influence.

PubMed

Nielfa, A; Cano, R; Pérez, A; Fdez-Polanco, M

2015-03-01

Solid wastes from industrial, commercial and community activities are of growing concern as the total volume of waste produced continues to increase. The knowledge of the specific composition and characteristics of the waste is an important tool in the correct development of the anaerobic digestion process. The problems derived from the anaerobic digestion of sole substrates with high lipid, carbohydrate or protein content lead to the co-digestion of these substrates with another disposed waste, such as sewage sludge. The kinetic of the anaerobic digestion is especially difficult to explain adequately, although some mathematical models are able to represent the main aspects of a biological system, thus improving understanding of the parameters involved in the process. The aim of this work is to evaluate the experimental biochemical methane potential on the co-digestion of sewage sludge with different solid wastes (grease; spent grain and cow manure) through the implementation of four kinetic models. The co-digestion of grease waste and mixed sludge obtained the best improvements from the sole substrates, with additional positive synergistic effects. The Gompertz model fits the experimental biochemical methane potential to an accuracy of 99%, showing a correlation between the percentage of lipid in the substrates and co-digestions and the period of lag phase. © The Author(s) 2015.

Modeling of the interaction between osteoblasts and biocompatible substrates as a function of adhesion strength.

PubMed

Portan, D V; Deligianni, D D; Deligianni, K; Kroustalli, A A; Tyllianakis, M; Papanicolaou, G C

2018-03-01

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. Nanoscale structured substrates [e.g., titania nanotubes (TNTs) or carbon nanotubes (CNTs)] dramatically improve the functions of conventional biomaterials. The present investigation evaluated the behavior of osteoblasts cells cultured on smooth and nanostructured substrates, by measuring osteoblasts specific biomarkers [alkaline phosphatase (AP) and total protein] and cells adhesion strength to substrates, followed by semi-empirical modeling to predict the experimental results. Findings were in total agreement with the current state of the art. The proliferation, as well as the AP and total protein levels were higher on the nanostructure phases (TNTs, CNTs) comparing to the smooth ones (plastic and pure titanium). Cells adhesion strength measured was found higher on the nanostructured materials. This coincided with a higher value of proteins which are directly implicated in the process of adherence. Results were accurately predicted through the Viscoelastic Hybrid Interphase Model. A gradual adherence of bone cells to implants using multilayered biomaterials that involve biodegradable polymeric films and a nanoscale modification of titanium surface is suggested to improve performance through an interphase-mediated osteointegration of orthopedic implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 621-628, 2018. © 2017 Wiley Periodicals, Inc.

Effect of specific surface microstructures on substrate endothelialisation and thrombogenicity: Importance for stent design.

PubMed

Lutter, Christoph; Nothhaft, Matthias; Rzany, Alexander; Garlichs, Christoph D; Cicha, Iwona

2015-01-01

In coronary artery disease, highly stenosed arteries are frequently treated by stent implantation, which thereafter necessitates a dual-antiplatelet therapy (DAPT) in order to prevent stent-thrombosis. We hypothesized that specific patterns of microstructures on stents can accelerate endothelialisation thereby reducing their thrombogenicity and the DAPT duration. Differently designed, 2-5 μm high elevations or hollows were lithographically etched on silicon plates, subsequently coated with silicon carbide. Smooth silicon plates and bare metal substrates were used as controls. To assess attachment and growth of human umbilical vein endothelial cells under static or flow conditions, actin cytoskeleton was visualised with green phalloidin. Endothelial migration was assessed in a modified barrier assay. To investigate surface thrombogenicity, platelets were incubated on the structured surfaces in static and flow conditions, and visualised with fluorescein-conjugated P-selectin antibody. Images were taken with incident-light fluorescent microscope for non-transparent objects. Compared to smooth surface, flat cubic elevations (5 μm edge length) improved endothelial cell attachment and growth under static and dynamic conditions, whereas smaller, spiky structures (2 μm edge length) had a negative influence on endothelialisation. Endothelial cell migration was fastest on flat cubic elevations, hollows, and smooth surfaces, whereas spiky structures and bare metal had a negative effect on endothelial migration. Thrombogenicity assays under static and flow conditions showed that platelet adhesion was reduced on the flat elevations and the smooth surface, as compared to the spiky structures, the hollow design and the bare metal substrates. Surface microstructures strongly influence endothelialisation of substrates. Designing stents with surface topography which accelerates endothelialisation and reduces thrombogenicity may be of clinical benefit by improving the safety profile of coronary interventions.

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Modular and selective biosynthesis of gasoline-range alkanes.

PubMed

Sheppard, Micah J; Kunjapur, Aditya M; Prather, Kristala L J

2016-01-01

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

PubMed Central

Ramirez, Monica L. Gonzalez; Poreba, Marcin; Snipas, Scott J.; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S.

2018-01-01

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. PMID:29414788

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

PubMed

Juettner, Norbert E; Schmelz, Stefan; Bogen, Jan P; Happel, Dominic; Fessner, Wolf-Dieter; Pfeifer, Felicitas; Fuchsbauer, Hans-Lothar; Scrima, Andrea

2018-05-01

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI p ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI p exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPI p accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications. © 2018 The Protein Society.

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

PubMed

Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

2016-05-09

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Notch-modifying xylosyltransferase-substrate complexes support an SNi-like retaining mechanism

PubMed Central

Yu, Hongjun; Takeuchi, Megumi; LeBarron, Jamie; Kantharia, Joshua; London, Erwin; Bakker, Hans; Haltiwanger, Robert S.; Li, Huilin; Takeuchi, Hideyuki

2015-01-01

A major remaining question in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xylosideα1–3 Xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly the Epidermal Growth Factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations where dysregulation of Notch is known to cause cancer or developmental disorders. PMID:26414444

Substrate milling pretreatment as a key parameter for Solid-State Anaerobic Digestion optimization.

PubMed

Motte, J-C; Escudié, R; Hamelin, J; Steyer, J-P; Bernet, N; Delgenes, J-P; Dumas, C

2014-12-01

The effect of milling pretreatment on performances of Solid-State Anaerobic Digestion (SS-AD) of raw lignocellulosic residue is still controverted. Three batch reactors treating different straw particle sizes (milled 0.25 mm, 1 mm and 10 mm) were followed during 62 days (6 sampling dates). Although a fine milling improves substrate accessibility and conversion rate (up to 30% compared to coarse milling), it also increases the risk of media acidification because of rapid and high acids production during fermentation of the substrate soluble fraction. Meanwhile, a gradual adaptation of microbial communities, were observed according to both reaction progress and methanogenic performances. The study concluded that particle size reduction affected strongly the performances of the reaction due to an increase of substrate bioaccessibility. An optimization of SS-AD processes thanks to particle size reduction could therefore be applied at farm or industrial scale only if a specific management of the soluble compounds is established. Copyright © 2014 Elsevier Ltd. All rights reserved.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

2016-01-01

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, F.H.; Moore, J.C.

1999-05-25

A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, F.H.; Moore, J.C.

1998-04-21

A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases. These enzymes exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

PubMed

Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Partial Reconstruction of Flavonoid and Isoflavonoid Biosynthesis in Yeast Using Soybean Type I and Type II Chalcone Isomerases1[w

PubMed Central

Ralston, Lyle; Subramanian, Senthil; Matsuno, Michiyo; Yu, Oliver

2005-01-01

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux. PMID:15778463

Characterizing Protease Specificity: How Many Substrates Do We Need?

PubMed Central

Schauperl, Michael; Fuchs, Julian E.; Waldner, Birgit J.; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4’) with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design. PMID:26559682

Paramagnetic iron-doped hydroxyapatite nanoparticles with improved metal sorption properties. A bioorganic substrates-mediated synthesis.

PubMed

Mercado, D Fabio; Magnacca, Giuliana; Malandrino, Mery; Rubert, Aldo; Montoneri, Enzo; Celi, Luisella; Bianco Prevot, Alessandra; Gonzalez, Mónica C

2014-03-26

This paper describes the synthesis of paramegnetic iron-containing hydroxyapatite nanoparticles and their increased Cu(2+) sorbent capacity when using Ca(2+) complexes of soluble bioorganic substrates from urban wastes as synthesis precursors. A thorough characterization of the particles by TEM, XRD, FTIR spectroscopy, specific surface area, TGA, XPS, and DLS indicates that loss of crystallinity, a higher specific area, an increased surface oxygen content, and formation of surface iron phases strongly enhance Cu(2+) adsorption capacity of hydroxyapatite-based materials. However, the major effect of the surface and morphologycal modifications is the size diminution of the aggregates formed in aqueous solutions leading to an increased effective surface available for Cu(2+) adsorption. Maximum sorption values of 550-850 mg Cu(2+) per gram of particles suspended in an aqueous solution at pH 7 were determined, almost 10 times the maximum values observed for hydroxyapatite nanoparticles suspensions under the same conditions.

Design of the hairpin ribozyme for targeting specific RNA sequences.

PubMed

Hampel, A; DeYoung, M B; Galasinski, S; Siwkowski, A

1997-01-01

The following steps should be taken when designing the hairpin ribozyme to cleave a specific target sequence: 1. Select a target sequence containing BN*GUC where B is C, G, or U. 2. Select the target sequence in areas least likely to have extensive interfering structure. 3. Design the conventional hairpin ribozyme as shown in Fig. 1, such that it can form a 4 bp helix 2 and helix 1 lengths up to 10 bp. 4. Synthesize this ribozyme from single-stranded DNA templates with a double-stranded T7 promoter. 5. Prepare a series of short substrates capable of forming a range of helix 1 lengths of 5-10 bp. 6. Identify these by direct RNA sequencing. 7. Assay the extent of cleavage of each substrate to identify the optimal length of helix 1. 8. Prepare the hairpin tetraloop ribozyme to determine if catalytic efficiency can be improved.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

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

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar tomore » tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.« less

Modification of the surface properties of glass-ceramic materials at low-pressure RF plasma stream

NASA Astrophysics Data System (ADS)

Tovstopyat, Alexander; Gafarov, Ildar; Galeev, Vadim; Azarova, Valentina; Golyaeva, Anastasia

2018-05-01

The surface roughness has a huge effect on the mechanical, optical, and electronic properties of materials. In modern optical systems, the specifications for the surface accuracy and smoothness of substrates are becoming even more stringent. Commercially available pre-polished glass-ceramic substrates were treated with the radio frequency (RF) inductively coupled (13.56 MHz) low-pressure plasma to clean the surface of the samples and decrease the roughness. Optical emission spectroscopy was used to investigate the plasma stream parameters and phase-shifted interferometry to investigate the surface of the specimen. In this work, the dependence of RF inductively coupled plasma on macroscopic parameters was investigated with the focus on improving the surfaces. The ion energy, sputtering rate, and homogeneity were investigated. The improvements of the glass-ceramic surfaces from 2.6 to 2.2 Å root mean square by removing the "waste" after the previous operations had been achieved.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

PubMed

Ramirez, Monica L Gonzalez; Poreba, Marcin; Snipas, Scott J; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S

2018-05-04

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A co-culture device with a tunable stiffness to understand combinatorial cell-cell and cell-matrix interactions.

PubMed

Rao, Nikhil; Grover, Gregory N; Vincent, Ludovic G; Evans, Samantha C; Choi, Yu Suk; Spencer, Katrina H; Hui, Elliot E; Engler, Adam J; Christman, Karen L

2013-11-01

Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

PubMed Central

Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin

2017-01-01

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo

PubMed Central

Shazeeb, Mohammed S.; Xie, Yang; Gupta, Suresh; Bogdanov, Alexei A.

2013-01-01

Bis-phenylamides and bis-hydroxyindolamides of DTPA(Gd) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, bis-5HT-DTPA(Gd) has been used to image localized inflammation in animal models by detecting neutrophil derived myeloperoxidase (MPO) activity at the inflammation site. However, in other pre-clinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here, we report a novel MPO sensing probe obtained by replacing the reducing substrate serotonin (5HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using NMR spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd): 1) improves solubility in water; 2) acts as a substrate for both HRP and MPO enzymes; 3) induces cross linking of proteins in the presence of MPO; 4) produces oxidation products which bind to plasma proteins and; 5) unlike bis-5HT-DTPA(Gd), does not follow first order reaction kinetics. In vivo MR imaging in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to five days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. In conclusion, bis-HTrp-DTPA(Gd) should offer improvements for MR imaging of MPO-mediated inflammation in vivo especially in high-field MRI, which requires higher dose of contrast agent. PMID:22954188

Hairy and Slippery Polyoxazoline-Based Copolymers on Model and Cartilage Surfaces.

PubMed

Morgese, Giulia; Ramakrishna, Shivaprakash N; Simic, Rok; Zenobi-Wong, Marcy; Benetti, Edmondo M

2018-02-12

Comb-like polymers presenting a hydroxybenzaldehyde (HBA)-functionalized poly(glutamic acid) (PGA) backbone and poly(2-methyl-2-oxazoline) (PMOXA) side chains chemisorb on aminolized substrates, including cartilage surfaces, forming layers that reduce protein contamination and provide lubrication. The structure, physicochemical, biopassive, and tribological properties of PGA-PMOXA-HBA films are finely determined by the copolymer architecture, its reactivity toward the surface, i.e. PMOXA side-chain crowding and HBA density, and by the copolymer solution concentration during assembly. Highly reactive species with low PMOXA content form inhomogeneous layers due to the limited possibility of surface rearrangements by strongly anchored copolymers, just partially protecting the functionalized surface from protein contamination and providing a relatively weak lubrication on cartilage. Biopassivity and lubrication can be improved by increasing copolymer concentration during assembly, leading to a progressive saturation of surface defects across the films. In a different way, less reactive copolymers presenting high PMOXA side-chain densities form uniform, biopassive, and lubricious films, both on model aminolized silicon oxide surfaces, as well as on cartilage substrates. When assembled at low concentrations these copolymers adopt a "lying down" conformation, i.e. adhering via their backbones onto the substrates, while at high concentrations they undergo a conformational transition, assuming a more densely packed, "standing up" structure, where they stretch perpendicularly from the substrate. This specific arrangement reduces protein contamination and improves lubrication both on model as well as on cartilage surfaces.

Comparative study on polyvinyl chloride film as flexible substrate for preparing free-standing polyaniline-based composite electrodes for supercapacitors.

PubMed

Wang, Hongxing; Liu, Dong; Du, Pengcheng; Wei, Wenli; Wang, Qi; Liu, Peng

2017-11-15

The free-standing polyaniline (PANI)-based composite film electrodes were prepared with polyvinyl chloride (PVC) and the aniline modified PVC (PVC-An) films as flexible substrates for supercapacitors, via facile in-situ chemical oxidative polymerization of aniline, with conventional chemical oxidative polymerization or rapid-mixing chemical oxidative polymerization technique. Owing to the grafting of PANI from the PVC-An film as substrate and the suppression of the secondary growth of the primary PANI particles in the rapid-mixing chemical oxidative polymerization, the PVC-g-PANI-2 composite film with loose surface possessed better comprehensive performance, accompanying the high specific capacitance (645.3F/g at a current density of 1A/g), good rate capacitance (retaining 63.2% of original value at a current density of 10A/g and 52.0% at a scan rate of 100mV/s), good cycle stability (retaining 83.1% after 1000 cycles) and the improved internal resistance. Besides its excellent flexibility, it could retain 61.2% of its original specific capacitance under the stress of 8.66MPa for 1h, demonstrating a good tensile-resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides.

PubMed

Geng, Xueran; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

2015-07-24

An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

Induced-fit Mechanism for Prolyl Endopeptidase

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

Li, Min; Chen, Changqing; Davies, David R.

2010-11-15

Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the {beta}-propeller and {alpha}/{beta}-hydrolase domains; addition of substrate tomore » preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.« less

Heterologous laccase production and its role in industrial applications

PubMed Central

Pezzella, Cinzia; Giardina, Paola; Faraco, Vincenza; Sannia, Giovanni

2010-01-01

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry. PMID:21327057

Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

PubMed

Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

2016-11-01

Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of a biomimetic 3D substrate based on the Human Elastin-like Polypeptides (HELPs) model system for elastolytic activity detection.

PubMed

Corich, Lucia; Busetti, Marina; Petix, Vincenzo; Passamonti, Sabina; Bandiera, Antonella

2017-08-10

Elastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli. Copyright © 2017 Elsevier B.V. All rights reserved.

Signal processing for the detection of explosive residues on varying substrates using laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Morton, Kenneth D., Jr.; Torrione, Peter A.; Collins, Leslie

2011-05-01

Laser induced breakdown spectroscopy (LIBS) can provide rapid, minimally destructive, chemical analysis of substances with the benefit of little to no sample preparation. Therefore, LIBS is a viable technology for the detection of substances of interest in near real-time fielded remote sensing scenarios. Of particular interest to military and security operations is the detection of explosive residues on various surfaces. It has been demonstrated that LIBS is capable of detecting such residues, however, the surface or substrate on which the residue is present can alter the observed spectra. Standard chemometric techniques such as principal components analysis and partial least squares discriminant analysis have previously been applied to explosive residue detection, however, the classification techniques developed on such data perform best against residue/substrate pairs that were included in model training but do not perform well when the residue/substrate pairs are not in the training set. Specifically residues in the training set may not be correctly detected if they are presented on a previously unseen substrate. In this work, we explicitly model LIBS spectra resulting from the residue and substrate to attempt to separate the response from each of the two components. This separation process is performed jointly with classifier design to ensure that the classifier that is developed is able to detect residues of interest without being confused by variations in the substrates. We demonstrate that the proposed classification algorithm provides improved robustness to variations in substrate compared to standard chemometric techniques for residue detection.

Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

NASA Astrophysics Data System (ADS)

Zhang, Yuanxing

Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

Substrate optimization and clinical validation of reporter peptides for MS-based protease profiling in serum specimens: a new approach for diagnosis of malignant disease.

PubMed

Yepes, Diego; Jacob, Anette; Dauber, Marc; Costina, Victor; Hofheinz, Ralf; Neumaier, Michael; Findeisen, Peter

2011-07-01

The progression of many solid tumors is characterized by the release of tumor-associated proteases, such as cancer procoagulant, MMP2 and MMP7. Consequently, the detection of tumor-specific proteolytic activity in serum specimens has recently been proposed as a new diagnostic tool in oncology. However, tumor-associated proteases are highly diluted in serum specimens and it is challenging to identify substrates that are specifically cleaved. In this study, we describe the systematic optimization of a synthetic peptide substrate using a positional scanning synthetic combinatorial library (PS-SCL) approach. The initial reporter peptide (RP) comprises of the cleavage site, WKPYDAAD, that is part of the coagulation factor X, the natural substrate of the tumor-associated cysteine protease cancer procoagulant (EC 3.4.22.26). Specifically, the amino acid substitution of aspartatic acid (D) in position P1' against asparagine (N) improved the processing of respective RPs in serum specimens from patients with colorectal tumors compared to healthy controls. Proteolytic fragments of RPs accumulated during prolonged incubation with serum specimens and were quantified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Finally, the optimized RP with the cleaved motif WKPYNAAD was combined with the RPs, VPLSLTMG and IPVSLRSG, that were cleaved by the tumor-associated proteases, MMP2 and MMP7, respectively. The diagnostic accuracy of MS-based protease profiling was evaluated for this triplex RP mix in a cohort of 50 serum specimens equally divided into colorectal cancer patients and healthy control individuals. Multiparametric analysis showed an AUC value of 0.90 for the receiver operating characteristic curve and was superior to the classification accuracy of the single markers. Our results demonstrate that RPs for MS-based protease profiling can systematically be optimized with a PS-SCL. Furthermore, the combination of different RPs can additionally increase the classification accuracy of functional protease profiling, and this in turn could lead to improved diagnosis, monitoring and prognosis of malignant disease.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors.

PubMed

de Veer, Simon J; Swedberg, Joakim E; Brattsand, Maria; Clements, Judith A; Harris, Jonathan M

2016-12-01

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki=4.2±0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.

High-quality substrate for fluorescence enhancement using agarose-coated silica opal film.

PubMed

Xu, Ming; Li, Juan; Sun, Liguo; Zhao, Yuanjin; Xie, Zhuoying; Lv, Linli; Zhao, Xiangwei; Xiao, Pengfeng; Hu, Jing; Lv, Mei; Gu, Zhongze

2010-08-01

To improve the sensitivity of fluorescence detection in biochip, a new kind of substrates was developed by agarose coating on silica opal film. In this study, silica opal film was fabricated on glass substrate using the vertical deposition technique. It can provide stronger fluorescence signals and thus improve the detection sensitivity. After coating with agarose, the hybrid film could provide a 3D support for immobilizing sample. Comparing with agarose-coated glass substrate, the agarose-coated opal substrates could selectively enhance particular fluorescence signals with high sensitivity when the stop band of the silica opal film in the agarose-coated opal substrate overlapped the fluorescence emission wavelength. A DNA hybridization experiment demonstrated that fluorescence intensity of special type of agarose-coated opal substrates was about four times that of agarose-coated glass substrate. These results indicate that the optimized agarose-coated opal substrate can be used for improving the sensitivity of fluorescence detection with high quality and selectivity.

Enhanced detection of type C botulinum neurotoxin by the Endopep-MS assay through optimization of peptide substrates

PubMed Central

Wang, Dongxia; Krilich, Joan; Baudys, Jakub; Barr, John R.; Kalb, Suzanne R.

2015-01-01

It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum neurotoxins, the most toxic substances and the causative agents of botulism. Type C botulinum neurotoxin (BoNT/C) represents one of the seven members of distinctive BoNT serotypes (A to G) that cause botulism in animals and avians. Here we report the development of optimized peptide substrates for improving the detection of BoNT/C and /CD mosaic toxins using an Endopep-MS assay, a mass spectrometry-based method that is able to rapidly and sensitively detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Based on the sequence of a short SNAP-25 peptide, we conducted optimization through a comprehensive process including length determination, terminal modification, single and multiple amino acid residue substitution, and incorporation of unnatural amino acid residues. Our data demonstrate that an optimal peptide provides a more than 200-fold improvement over the substrate currently used in the Endopep-MS assay for the detection of BoNT/C1 and /CD mosaic. Using the new substrate in a four-hour cleavage reaction, the limit of detection for the BoNT/C1 complex spiked in buffer, serum and milk samples was determined to be 0.5, 0.5 and 1 mouseLD50/mL, respectively, representing a similar or higher sensitivity than that obtained by traditional mouse bioassay. PMID:25913863

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

PubMed Central

Torres, Leticia L.; Cantero, Ángel; del Valle, Mercedes; Marina, Anabel; López-Gallego, Fernando; Guisán, José M.

2013-01-01

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the Km for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site. PMID:23263966

Effect of electric arc furnace slag on growth and physiology of maize (Zea mays L.).

PubMed

Radić, Sandra; Crnojević, Helena; Sandev, Dubravka; Jelić, Sonja; Sedlar, Zorana; Glavaš, Katarina; Pevalek-Kozlina, Branka

2013-12-01

Basic slag, used in this study as a potential source of certain nutrients, is a byproduct of the production of steel in electric arc furnace (EAF). A pot experiment with two nutrient-poor substrates was conducted to investigate to compare the effect of EAF steel slag and fertilizers NPK + F e on growth and availability of specific nutrients to maize. Mineral content of both substrate and plant leaves, growth, chlorophyll fluorescence and photosynthetic pigments were measured following six weeks of cultivation. As steel slag also contains trace amounts of heavy metals, certain oxidative parameters (antioxidative enzyme activities and lipid peroxidation) were evaluated as well. The steel slag improved soil mineral composition, increased above ground maize biomass by providing Fe, Mn, Mg, K and partly P and improved photosynthetic parameters. The potential phytotoxicity of EAF slag containing substrates was not determined as evaluated by MDA (malondialdehyde), GR (glutathione reductase) and APX (ascorbate peroxidase) levels. The obtained results show that EAF steel slag is comparable to NPK + F e in supplying nutrients for maize growth, indicating the potential of EAF steel slag as an inexpensive and non-phytotoxic nutrient supplier especially in poor soils.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

PubMed

Katsu, Kenjiro; Suzuki, Rintaro; Tsuchiya, Wataru; Inagaki, Noritoshi; Yamazaki, Toshimasa; Hisano, Tomomi; Yasui, Yasuo; Komori, Toshiyuki; Koshio, Motoyuki; Kubota, Seiji; Walker, Amanda R; Furukawa, Kiyoshi; Matsui, Katsuhiro

2017-12-11

Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F 2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE PAGES

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng; ...

2016-03-31

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

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

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

PubMed

Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

2012-04-27

Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

SynTec Final Technical Report: Synthetic biology for Tailored Enzyme cocktails

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

Lin, Janine; Teter, Sarah

Using a novel enzyme screening method inspired by synthetic biology, Novozymes developed new technology under SynTec which allows for more rapidly tailoring of enzyme cocktails. The methodology can be applied to specific feedstocks, and or coupled to address a specific hydrolytic conversion process context. Using combinatorial high throughput screening of libraries of enzyme domains, we can quickly assess which combination of catalytic modules delivers the best performance for a specific condition. To demonstrate the effectiveness of the screening process, we measured performance of the output catalytic cocktail compared to CTec3/HTec3. SynTec benchmark cocktail - blend of Cellic® CTec3 and HTec3.more » The test substrate was - ammonia fiber expansion pretreated corn stover (AFEX™ PCS).CTec3/HTec3 was assayed at the optimal pH and temperature, and also in the absence of any pH adjustment. The new enzyme cocktail discovered under SynTec was assayed in the absence of any pH adjustment and at the optimal temperature. Conversion is delivered by SynTec enzyme at significant dose reduction relative to CTec3/HTec3 at the controlled pH optimum, and without titrant required to maintain pH, which delivers additional cost savings relative to current state of the art process. In this 2.5 year $4M project, the team delivered an experimental cocktail that significantly outperformed CTec3/HTec3 for a specific substrate, and for specific hydrolysis conditions. As a means of comparing performance improvement delivered per research dollar spent, we note that SynTec delivered a similar performance improvement to the previous award, in a shorter time and with fewer resources than for the previously successful DOE project DECREASE, a 3.5 year, $25M project, though this project focused on a different substrate and used different hydrolysis conditions. The newly implemented technology for rapid sourcing of new cellulases and hemicellulases from nature is an example of Novozymes' continued innovation that results in more effective products for the advanced biofuel market.« less

Directions for Optimization of Photosynthetic Carbon Fixation: RuBisCO's Efficiency May Not Be So Constrained After All.

PubMed

Cummins, Peter L; Kannappan, Babu; Gready, Jill E

2018-01-01

The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate. Although the dominant hypothesis has been that the active-site chemistry constrains the enzyme's evolution, a more recent study on RuBisCO stability and adaptability has implicated competing selection pressures. Elucidating these constraints is crucial for directing future research on improving photosynthesis, as the current literature casts doubt on the potential effectiveness of site-directed mutagenesis to improve RuBisCO's efficiency. Here we use regression analysis to quantify the relationships between kinetic parameters obtained from empirical data sets spanning a wide evolutionary range of RuBisCOs. Most significantly we found that the rate constant for dissociation of CO 2 from the enzyme complex was much higher than previous estimates and comparable with the corresponding catalytic rate constant. Observed trends between relative specificity and turnover rate can be expressed as the product of negative and positive correlation factors. This provides an explanation in simple kinetic terms of both the natural variation of relative specificity as well as that obtained by reported site-directed mutagenesis results. We demonstrate that the kinetic behaviour shows a lesser rather than more constrained RuBisCO, consistent with growing empirical evidence of higher variability in relative specificity. In summary our analysis supports an explanation for the origin of the tradeoff between specificity and turnover as due to competition between protein stability and activity, rather than constraints between rate constants imposed by the underlying chemistry. Our analysis suggests that simultaneous improvement in both specificity and turnover rate of RuBisCO is possible.

Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

PubMed Central

Lee, Rachel S.; House, Colin M.; Cristiano, Briony E.; Hannan, Ross D.; Pearson, Richard B.; Hannan, Katherine M.

2011-01-01

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ. PMID:21869924

Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues

PubMed Central

Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine

2017-01-01

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. PMID:28877217

Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

PubMed

Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

2014-04-10

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Improving surface-enhanced Raman scattering effect using gold-coated hierarchical polystyrene bead substrates modified with postgrowth microwave treatment.

PubMed

Yuen, Clement; Zheng, Wei; Huang, Zhiwei

2008-01-01

We report a novel postgrowth microwave heating implementation by selectively modifying hierarchical polystyrene (PS) bead substrates coated with gold (Au) films to effectively improve the surface-enhanced Raman scattering (SERS) effect on the analytes. The SERS signal of probe molecule rhodamine 6G (Rh 6G) on the microwave-treated Au-PS substrates can be improved by 10-fold, while the detection limit of Rh 6G in concentration can be enhanced by two orders of magnitude compared to the as-growth substrates. The high-quality SERS spectrum of saliva can also be acquired using the modified substrates, demonstrating the potential for the realization of the high-performance SERS substrates for biomedical applications.

Improved conversion efficiency in dye-sensitized solar cells based on electrospun Al-doped ZnO nanofiber electrodes prepared by seed layer treatment

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

Yun Sining, E-mail: alexsyun1974@yahoo.com.c; Lim, Sangwoo

2011-02-15

The application of electrospun nanofibers in electronic devices is limited due to their poor adhesion to conductive substrates. To improve this, a seed layer (SD) is introduced on the FTO substrate before the deposition of the electrospun composite nanofibers. This facilitates the release of interfacial tensile stress during calcination and enhances the interfacial adhesion of the AZO nanofiber films with the FTO substrate. Dye-sensitized solar cells (DSSC) based on these AZO nanofiber photoelectrodes have been fabricated and investigated. An energy conversion efficiency ({eta}) of 0.54-0.55% has been obtained under irradiation of AM 1.5 simulated sunlight (100 mW/cm{sup 2}), indicating amore » massive improvement of {eta} in the AZO nanofiber film DSSCs after SD-treatment of the FTO substrate as compared to those with no treatment. The SD-treatment has been demonstrated to be a simple and facile method to solve the problem of poor adhesion between electrospun nanofibers and the conductive substrate. -- Graphical abstract: The poor adhesion between electrospun nanofibers and substrate is improved by a simple and facile seed layer (SD) treatment. The energy conversion efficiency of AZO nanofiber-based DSSCs has been greatly increased by SD-treatment of the FTO substrate. Display Omitted Research highlights: {yields} A simple and facile method (SD-treatment) has been demonstrated. {yields} The poor adhesion between electrospun nanofibers and substrate is improved by the SD-treatment. {yields} The {eta} of AZO nanofiber-based DSSCs has been greatly improved by SD-treatment of the FTO substrate.« less

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

PubMed Central

2012-01-01

Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development. PMID:22330134

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

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

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE PAGES

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...

2015-02-18

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Nanotextured PDMS Substrates for Enhanced Roughness and Aptamer Immobilization for Cancer Cell Capture

NASA Astrophysics Data System (ADS)

Islam, Muhymin; Mahmood, Arif; Bellah, Md.; Kim, Young-Tae; Iqbal, Samir

2014-03-01

Detection of circulating tumor cells (CTCs) in the early stages of cancer is requires very sensitive approach. Nanotextured polydimethylsiloxane (PDMS) substrates were fabricated by micro reactive ion etching (Micro-RIE) to have better control on surface morphology and to improve the affinity of PDMS surfaces to capture cancer cells using surface immobilized aptamers. The aptamers were specific to epidermal growth factor receptors (EGFR) present in cell membranes, and overexpressed in tumor cells. We also investigated the effect of nano-scale features on cell capturing by implementing various surfaces of different roughnesses. Three different recipes were used to prepare nanotextured PDMS by micro-RIE using oxygen (O2) and carbon tetrafluoride (CF4). The measured average roughness of three nanotextured PDMS surfaces were found to impact average densities of captured cells. In all cases, nanotextured PDMS facilitated cell capturing possibly due to increased effective surface area of roughened substrates at nanoscale. It was also observed that cell capture efficiency was higher for higher surface roughness. The nanotextured PDMS substrates are thus useful for cancer cytology devices.

Process for depositing hard coating in a nozzle orifice

DOEpatents

Flynn, P.L.; Giammarise, A.W.

1991-10-29

The present invention is directed to a process for coating the interior surfaces of an orifice in a substrate that forms a slurry fuel injection nozzle. In a specific embodiment, the nozzle is part of a fuel injection system for metering a coal-water slurry into a large, medium-speed, multi-cylinder diesel engine. In order to retard erosion of the orifice, the substrate is placed in a chemical vapor deposition (CVD) reaction chamber. A reaction gas is passed into the chamber at a gas temperature below its reaction temperature and is directed through the orifice in the substrate. The gas reaction temperature is a temperature at and above which the reaction gas deposits as a coating, and the reaction gas is of a composition whereby improved resistance to erosion by flow of the particulates in the slurry fuel is imparted by the deposited coating. Only the portion of the substrate in proximity to the orifice to be coated is selectively heated to at least the gas reaction temperature for effecting coating of the orifice's interior surfaces by the vapor deposited coating formed from the reaction gas. 2 figures.

Apparatus for depositing hard coating in a nozzle orifice

DOEpatents

Flynn, P.L.; Giammarise, A.W.

1995-02-21

The present invention is directed to a process for coating the interior surfaces of an orifice in a substrate that forms a slurry fuel injection nozzle. In a specific embodiment, the nozzle is part of a fuel injection system for metering a coal-water slurry into a large, medium-speed, multi-cylinder diesel engine. In order to retard erosion of the orifice, the substrate is placed in a chemical vapor deposition (CVD) reaction chamber. A reaction gas is passed into the chamber at a gas temperature below its reaction temperature and is directed through the orifice in the substrate. The gas reaction temperature is a temperature at and above which the reaction gas deposits as a coating, and the reaction gas is of a composition whereby improved resistance to erosion by flow of the particulates in the slurry fuel is imparted by the deposited coating. Only the portion of the substrate in proximity to the orifice to be coated is selectively heated to at least the gas reaction temperature for effecting coating of the orifice`s interior surfaces by the vapor deposited coating formed from the reaction gas. 2 figs.

Apparatus for depositing hard coating in a nozzle orifice

DOEpatents

Flynn, Paul L.; Giammarise, Anthony W.

1995-01-01

The present invention is directed to a process for coating the interior surfaces of an orifice in a substrate that forms a slurry fuel injection nozzle. In a specific embodiment, the nozzle is part of a fuel injection system for metering a coal-water slurry into a large, medium-speed, multi-cylinder diesel engine. In order to retard erosion of the orifice, the substrate is placed in a chemical vapor deposition (CVD) reaction chamber. A reaction gas is passed into the chamber at a gas temperature below its reaction temperature and is directed through the orifice in the substrate. The gas reaction temperature is a temperature at and above which the reaction gas deposits as a coating, and the reaction gas is of a composition whereby improved resistance to erosion by flow of the particulates in the slurry fuel is imparted by the deposited coating. Only the portion of the substrate in proximity to the orifice to be coated is selectively heated to at least the gas reaction temperature for effecting coating of the orifice's interior surfaces by the vapor deposited coating formed from the reaction gas.

Apparatus and process for depositing hard coating in a nozzle orifice

DOEpatents

Flynn, Paul L.; Giammarise, Anthony W.

1994-01-01

The present invention is directed to a process for coating the interior surfaces of an orifice in a substrate that forms a slurry fuel injection nozzle. In a specific embodiment, the nozzle is part of a fuel injection system for metering a coal-water slurry into a large, medium-speed, multi-cylinder diesel engine. In order to retard erosion of the orifice, the substrate is placed in a chemical vapor deposition (CVD) reaction chamber. A reaction gas is passed into the chamber at a gas temperature below its reaction temperature and is directed through the orifice in the substrate. The gas reaction temperature is a temperature at and above which the reaction gas deposits as a coating, and the reaction gas is of a composition whereby improved resistance to erosion by flow of the particulates in the slurry fuel is imparted by the deposited coating. Only the portion of the substrate in proximity to the orifice to be coated is selectively heated to at least the gas reaction temperature for effecting coating of the orifice's interior surfaces by the vapor deposited coating formed from the reaction gas.

Process for depositing hard coating in a nozzle orifice

DOEpatents

Flynn, Paul L.; Giammarise, Anthony W.

1991-01-01

The present invention is directed to a process for coating the interior surfaces of an orifice in a substrate that forms a slurry fuel injection nozzle. In a specific embodiment, the nozzle is part of a fuel injection system for metering a coal-water slurry into a large, medium-speed, multi-cylinder diesel engine. In order to retard erosion of the orifice, the substrate is placed in a chemical vapor deposition (CVD) reaction chamber. A reaction gas is passed into the chamber at a gas temperature below its reaction temperature and is directed through the orifice in the substrate. The gas reaction temperature is a temperature at and above which the reaction gas deposits as a coating, and the reaction gas is of a composition whereby improved resistance toerosion by flow of the particulates in the slurry fuel is imparted by the deposited coating. Only the portion of the substrate in proximity to the orifice to be coated is selectively heated to at least the gas reaction temperature for effecting coating of the orifice's interior surfaces by the vapor deposited coating formed from the reaction gas.

Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes.

PubMed

Kumar, Rahul

2016-01-01

Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species.

Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells

PubMed Central

Wasnik, Samiksha; Kantipudi, Suma; Kirkland, Mark A.; Pande, Gopal

2016-01-01

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion. PMID:26981135

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis.

PubMed

Stiti, Naim; Missihoun, Tagnon D; Kotchoni, Simeon O; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected ArabidopsisALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

A new approach to the immobilisation of poly(ethylene oxide) for the reduction of non-specific protein adsorption on conductive substrates

NASA Astrophysics Data System (ADS)

Cole, Martin A.; Thissen, Helmut; Losic, Dusan; Voelcker, Nicolas H.

2007-04-01

Biomedical and biotechnological devices often require surface modifications to improve their performance. In most cases, uniform coatings are desired which provide a specific property or lead to a specific biological response. In the present work, we have generated pinhole-free coatings providing amine functional groups achieved by electropolymerisation of tyramine on highly doped silicon substrates. Furthermore, amine groups were used for the subsequent grafting of poly(ethylene oxide) aldehyde via reductive amination. All surface modification steps were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The results indicate that the stability and the density of amine functional groups introduced at the surface via electropolymerisation compare favourably with alternative coatings frequently used in biomedical and biotechnological devices such as plasma polymer films. Furthermore, protein adsorption on amine and poly(ethylene oxide) coatings was studied by XPS and a colorimetric assay to test enzymatic activity. The grafting of poly(ethylene oxide) under cloud point conditions on electropolymerised tyramine layers resulted in surfaces with extremely low protein fouling character.

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis

PubMed Central

Stiti, Naim; Missihoun, Tagnon D.; Kotchoni, Simeon O.; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected Arabidopsis ALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities. PMID:22639603

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

PubMed Central

Song, Ling

2011-01-01

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division. PMID:21191176

Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

PubMed

Damle, Nikhil Prakash; Mohanty, Debasisa

2014-06-15

In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

Substrate Sorting by a Supercharged Nanoreactor

PubMed Central

2017-01-01

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts. PMID:29278496

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

Engineering Cellulases for Biorefinery

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

Kumar, Manoj

2010-06-27

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Thermostable Cellulases: Why & How?

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

Kumar, Manoj

2010-04-19

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Cell foundry with high product specificity and catalytic activity for 21-deoxycortisol biotransformation.

PubMed

Xiong, Shuting; Wang, Ying; Yao, Mingdong; Liu, Hong; Zhou, Xiao; Xiao, Wenhai; Yuan, Yingjin

2017-06-13

21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity. In this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known. Heterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.

Developing Enzyme and Biomimetic Catalysts for Upgrading Heavy Crudes via Biological Hydrogenation and Hydrodesulfurization

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

Borole, A P

The recovery and conversion of heavy oils is limited due to the high viscosity of these crudes and their high heteroatom content. Conventional technology relies on thermochemical hydrogenation and hydrodesulfurization to address these problems and is energy intensive due to the high operating temperature and pressure. This project was initiated to explore biological catalysts for adding hydrogen to the heavy oil molecules. Biological enzymes are efficient at hydrogen splitting at very mild conditions such as room temperature and pressure, however, they are very specific in terms of the substrates they hydrogenate. The goal of the project was to investigate howmore » the specificity of these enzymes can be altered to develop catalysts for oil upgrading. Three approaches were used. First was to perform chemical modification of the enzyme surface to improve binding of other non-natural substrates. Second approach was to expose the deeply buried catalytic active site of the enzyme by removal of protein scaffolding to enable better interaction with other substrates. The third approach was based on molecular biology to develop genetically engineered systems for enabling targeted structural changes in the enzyme. The first approach was found to be limited in success due to the non-specificity of the chemical modification and inability to target the region near the active site or the site of substrate binding. The second approach produced a smaller catalyst capable of catalyzing hydrogen splitting, however, further experimentation is needed to address reproducibility and stability issues. The third approach which targeted cloning of hydrogenase in alternate hosts demonstrated progress, although further work is necessary to complete the cloning process. The complex nature of the hydrogenase enzyme structure-function relationship and role of various ligands in the protein require significant more research to better understand the enzyme and to enable success in strategies in developing catalysts with broader specificity as that required for crude upgrading.« less

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion-controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity.

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

PubMed Central

Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

2013-01-01

SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

PubMed

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

PubMed Central

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532

Improved specific productivity in cephalexin synthesis by immobilized PGA in silica magnetic micro-particles.

PubMed

Bernardino, Susana M S A; Fernandes, Pedro; Fonseca, Luís P

2010-12-01

There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by "green" alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties was performed in aqueous media with PGME and 7-ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7-ADCA ratio of 3 was considered optimum. A maximum specific productivity of 5.9 mmol h(-1), gbiocatalyst(-1) at 160 mM 7-ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol(-1) 7-ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half-life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7-ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin. © 2010 Wiley Periodicals, Inc.

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Ultra-slim flexible glass for roll-to-roll electronic device fabrication

NASA Astrophysics Data System (ADS)

Garner, Sean; Glaesemann, Scott; Li, Xinghua

2014-08-01

As displays and electronics evolve to become lighter, thinner, and more flexible, the choice of substrate continues to be critical to their overall optimization. The substrate directly affects improvements in the designs, materials, fabrication processes, and performance of advanced electronics. With their inherent benefits such as surface quality, optical transmission, hermeticity, and thermal and dimensional stability, glass substrates enable high-quality and long-life devices. As substrate thicknesses are reduced below 200 μm, ultra-slim flexible glass continues to provide these inherent benefits to high-performance flexible electronics such as displays, touch sensors, photovoltaics, and lighting. In addition, the reduction in glass thickness also allows for new device designs and high-throughput, continuous manufacturing enabled by R2R processes. This paper provides an overview of ultra-slim flexible glass substrates and how they enable flexible electronic device optimization. Specific focus is put on flexible glass' mechanical reliability. For this, a combination of substrate design and process optimizations has been demonstrated that enables R2R device fabrication on flexible glass. Demonstrations of R2R flexible glass processes such as vacuum deposition, photolithography, laser patterning, screen printing, slot die coating, and lamination have been made. Compatibility with these key process steps has resulted in the first demonstration of a fully functional flexible glass device fabricated completely using R2R processes.

Substrate thermal conductivity effect on heat dissipation and lifetime improvement of organic light-emitting diodes

NASA Astrophysics Data System (ADS)

Chung, Seungjun; Lee, Jae-Hyun; Jeong, Jaewook; Kim, Jang-Joo; Hong, Yongtaek

2009-06-01

We report substrate thermal conductivity effect on heat dissipation and lifetime improvement of organic light-emitting diodes (OLEDs). Heat dissipation behavior of top-emission OLEDs fabricated on silicon, glass, and planarized stainless steel substrates was measured by using an infrared camera. Peak temperature measured from the backside of each substrate was saturated to be 21.4, 64.5, and 40.5 °C, 180 s after the OLED was operated at luminance of 10 000 cd/m2 and 80% luminance lifetime was about 198, 31, and 96 h, respectively. Efficient heat dissipation through the highly thermally conductive substrates reduced temperature increase, resulting in much improved OLED lifetime.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer.

PubMed

Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi

2015-04-15

In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*

PubMed Central

Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.

2014-01-01

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo.

PubMed

Shazeeb, Mohammed S; Xie, Yang; Gupta, Suresh; Bogdanov, Alexei A

2012-01-01

Bis-phenylamides and bis-hydroxyindolamides of diethylenetriaminepentaacetic acid-gadolinium (DTPA(Gd)) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, gadolinium chelates of bis-5-hydroxytryptamide-DTPA (bis-5HT-DTPA(Gd)) have been used to image localized inflammation in animal models by detecting neutrophil-derived myeloperoxidase (MPO) activity at the inflammation site. However, in other preclinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here we report a novel MPO-sensing probe obtained by replacing the reducing substrate serotonin (5-HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using nuclear magnetic resonance spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd) (1) improves solubility in water; (2) acts as a substrate for both horseradish peroxidase and MPO enzymes; (3) induces cross-linking of proteins in the presence of MPO; (4) produces oxidation products, which bind to plasma proteins; and (5) unlike bis-5HT-DTPA(Gd), does not follow first-order reaction kinetics. In vivo magnetic resonance imaging (MRI) in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to 5 days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. Bis-HTrp-DTPA(Gd) should offer improvements for MRI of MPO-mediated inflammation in vivo, especially in high-field MRI, which requires a higher dose of contrast agent.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

A study to improve the mechanical properties of silicon carbide ribbon fibers

NASA Technical Reports Server (NTRS)

Debolt, H. E.; Robey, R. J.

1976-01-01

Preliminary deposition studies of SiC ribbon on a carbon ribbon substrate showed that the dominant strength limiting flaws were at the substrate surface. Procedures for making the carbon ribbon substrate from polyimide film were improved, providing lengths up to 450 meters (1,500 ft.) of flat carbon ribbon substrate 1,900 microns (75 mils) wide by 25 microns (1 mil) thick. The flaws on the carbon ribbon were smaller and less frequent than on carbon ribbon used earlier. SiC ribbon made using the improved substrate, including a layer of pyrolytic graphite to reduce further the severity of substrate surface flaws, showed strength levels up to the 2,068 MPa (300 Ksi) target of the program, with average strength levels over 1,700 MPa (250 Ksi) with coefficient of variation as low as 10% for some runs.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa.

PubMed

Gao, Chongliang; Lan, Dongming; Liu, Lu; Zhang, Houjin; Yang, Bo; Wang, Yonghua

2014-07-01

The lipase from Malassezia globosa (SMG1) has specific activity on mono- and diacylglycerol but not on triacylglycerol. The structural analysis of SMG1 structure shows that two bulky aromatic residues, W116 and W229, lie at the entrance of the active site. To study the functions of these two residues in the substrate recognition and the catalytic reaction, they were mutated to a series of amino acids. Subsequently, biochemical properties of these mutants were investigated. Although the activities decrease, W229L and W116A show a significant shift in substrate preference. W229L has an increased preference for short-chain substrates whereas W116A has preference for long-chain substrates. Besides, the half-lives of W116A and W116H at 45 °C are 346.6 min and 115.5 min respectively, which improve significantly compared to that of native enzyme. Moreover, the optimum substrate of W116A, W116F and W229F mutants shifted from p-nitrophenyl caprylate to p-nitrophenyl myristate. These findings not only shed light onto the lipase structure/function relationship but also lay the framework for the potential industrial applications. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A framework for understanding shared substrates of airway protection

PubMed Central

TROCHE, Michelle Shevon; BRANDIMORE, Alexandra Essman; GODOY, Juliana; HEGLAND, Karen Wheeler

2014-01-01

Deficits of airway protection can have deleterious effects to health and quality of life. Effective airway protection requires a continuum of behaviors including swallowing and cough. Swallowing prevents material from entering the airway and coughing ejects endogenous material from the airway. There is significant overlap between the control mechanisms for swallowing and cough. In this review we will present the existing literature to support a novel framework for understanding shared substrates of airway protection. This framework was originally adapted from Eccles' model of cough28 (2009) by Hegland, et al.42 (2012). It will serve to provide a basis from which to develop future studies and test specific hypotheses that advance our field and ultimately improve outcomes for people with airway protective deficits. PMID:25141195

Quantum dot-based microfluidic biosensor for cancer detection

NASA Astrophysics Data System (ADS)

Ghrera, Aditya Sharma; Pandey, Chandra Mouli; Ali, Md. Azahar; Malhotra, Bansi Dhar

2015-05-01

We report results of the studies relating to fabrication of an impedimetric microfluidic-based nucleic acid sensor for quantification of DNA sequences specific to chronic myelogenous leukemia (CML). The sensor chip is prepared by patterning an indium-tin-oxide (ITO) coated glass substrate via wet chemical etching method followed by sealing with polydimethylsiloxane (PDMS) microchannel for fluid control. The fabricated microfluidic chip comprising of a patterned ITO substrate is modified by depositing cadmium selenide quantum dots (QCdSe) via Langmuir-Blodgett technique. Further, the QCdSe surface has been functionalized with specific DNA probe for CML detection. The probe DNA functionalized QCdSe integrated miniaturized system has been used to monitor target complementary DNA concentration by measuring the interfacial charge transfer resistance via hybridization. The presence of complementary DNA in buffer solution significantly results in decreased electro-conductivity of the interface due to presence of a charge barrier for transport of the redox probe ions. The microfluidic DNA biosensor exhibits improved linearity in the concentration range of 10-15 M to 10-11 M.

Insight into the substrate specificity change caused by the Y227H mutation of α-glucosidase III from the European honeybee (Apis mellifera) through molecular dynamics simulations.

PubMed

Na Ayutthaya, Pratchaya Pramoj; Chanchao, Chanpen; Chunsrivirot, Surasak

2018-01-01

Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

High-yield production of mannitol by Leuconostoc pseudomesenteroides CTCC G123 from chicory-derived inulin hydrolysate.

PubMed

Zhang, Min; Gu, Lei; Cheng, Chao; Zhu, Junru; Wu, Hao; Ma, Jiangfeng; Dong, Weiliang; Kong, Xiangping; Jiang, Min; Ouyang, Pingkai

2017-08-01

Chicory is an agricultural plant with considerable potential as a carbohydrate substrate for low-cost production of biochemicals. In this work, the production of mannitol by Leuconostoc pseudomesenteroides CTCC G123 from chicory-derived inulin hydrolysate was investigated. The bioconversion process initially suffered from the leakage of fructose to the phosphoketolase pathway, resulting in a low mannitol yield. When inulin hydrolysate was supplemented with glucose as a substrate for mannitol production in combination with aeration induction and nicotinic acid induced redox modulation strategies, the mannitol yield greatly improved. Under these conditions, significant improvement in the glucose consumption rate, intracellular NADH levels and mannitol dehydrogenase specific activity were observed, with mannitol production increasing from 64.6 to 88.1 g/L and overall yield increase from 0.69 to 0.94 g/g. This work demonstrated an efficient method for the production of mannitol from inulin hydrolysate with a high overall yield.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Improving the efficiency and directivity of THz photoconductive antennas by using a defective photonic crystal substrate

NASA Astrophysics Data System (ADS)

Rahmati, Ehsan; Ahmadi-Boroujeni, Mehdi

2018-04-01

One of the shortcomings of photoconductive (PC) antennas in terahertz (THz) generation is low effective radiated power in the desirable direction. In this paper, we propose a defective photonic crystal (DPC) substrate consisting of a customized 2D array of air holes drilled into a solid substrate in order to improve the radiation characteristics of THz PC antennas. The effect of the proposed structure on the performance of a conventional THz PC antenna has been examined from several aspects including radiation efficiency, directivity, and field distribution. By comparing the radiation performance of the THz antenna on the proposed DPC substrate to that of the conventional solid substrate, it is shown that the proposed technique can significantly improve the efficiency and directivity of the THz PC antenna over a wide frequency range. It is achieved by reducing the amount of power coupled to the substrate surface waves and limiting the radiation in undesirable directions. In addition, it is found that the sensitivity of directivity to the substrate thickness is considerably decreased and the adverse Fabry-Perot effects of the thick substrate are reduced by the application of the proposed DPC substrate.

Predominant nonproductive substrate binding by fungal cellobiohydrolase I and implications for activity improvement.

PubMed

Rabinovich, Mikhail L; Melnik, Maria S; Herner, Mikhail L; Voznyi, Yakov V; Vasilchenko, Lilia G

2018-05-21

Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes 9 subsites for β-D-glucose units, 7 of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent k cat of HjCel7A at the hydrolysis of β-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) k cat /K m for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-β-cellotetraose (MUG 4 ). Interaction of substrates with the subsites -6 and -5 proximal to the non-conserved Gln101 residue in HjCel7A decreases K m,ap by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with K d ∼2-9 nM via CBM and CD that captures 6 terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG 4 is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding. This article is protected by copyright. All rights reserved.

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

Selective biotinylation of Neisseria meningitidis group B capsular polysaccharide and application in an improved ELISA for the detection of specific antibodies.

PubMed

Diaz Romero, J; Outschoorn, I

1993-03-15

A method is described for the selective biotinylation of meningococcal capsular polysaccharide from Neisseria meningitidis group B and its application to an enzyme-linked immunoabsorbent assay (ELISA) to detect specific antibodies by immobilization on streptavidin-coated microtiter wells. Capsular polysaccharide from Neisseria meningitidis B has been biotinylated by specific periodate oxidation of terminal residues and condensation of the resulting aldehydes with biotin hydrazide, using a spin-column technique in the intermediate purification steps. The ELISA was optimized employing an extended reaction time between the label alkaline phosphatase and its most common substrate, p-nitrophenyl phosphate, together with evaluation of blocking agents to minimize non-specific binding. Specificity was demonstrated by a direct competitive enzyme immunoassay (EIA).

Smart substrates: Making multi-chip modules smarter

NASA Astrophysics Data System (ADS)

Wunsch, T. F.; Treece, R. K.

1995-05-01

A novel multi-chip module (MCM) design and manufacturing methodology which utilizes active CMOS circuits in what is normally a passive substrate realizes the 'smart substrate' for use in highly testable, high reliability MCMS. The active devices are used to test the bare substrate, diagnose assembly errors or integrated circuit (IC) failures that require rework, and improve the testability of the final MCM assembly. A static random access memory (SRAM) MCM has been designed and fabricated in Sandia Microelectronics Development Laboratory in order to demonstrate the technical feasibility of this concept and to examine design and manufacturing issues which will ultimately determine the economic viability of this approach. The smart substrate memory MCM represents a first in MCM packaging. At the time the first modules were fabricated, no other company or MCM vendor had incorporated active devices in the substrate to improve manufacturability and testability, and thereby improve MCM reliability and reduce cost.

Remote magnetic navigation for accurate, real-time catheter positioning and ablation in cardiac electrophysiology procedures.

PubMed

Filgueiras-Rama, David; Estrada, Alejandro; Shachar, Josh; Castrejón, Sergio; Doiny, David; Ortega, Marta; Gang, Eli; Merino, José L

2013-04-21

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

PubMed Central

Filgueiras-Rama, David; Estrada, Alejandro; Shachar, Josh; Castrejón, Sergio; Doiny, David; Ortega, Marta; Gang, Eli; Merino, José L.

2013-01-01

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate. PMID:23628883

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

NASA Astrophysics Data System (ADS)

Sun, Yubing; Yong, Koh Meng Aw; Villa-Diaz, Luis G.; Zhang, Xiaoli; Chen, Weiqiang; Philson, Renee; Weng, Shinuo; Xu, Haoxing; Krebsbach, Paul H.; Fu, Jianping

2014-06-01

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Multifunctional potentiometric gas sensor array with an integrated temperature control and temperature sensors

DOEpatents

Blackburn, Bryan M; Wachsman, Eric D

2015-05-12

Embodiments of the subject invention relate to a gas sensor and method for sensing one or more gases. An embodiment incorporates an array of sensing electrodes maintained at similar or different temperatures, such that the sensitivity and species selectivity of the device can be fine tuned between different pairs of sensing electrodes. A specific embodiment pertains to a gas sensor array for monitoring combustion exhausts and/or chemical reaction byproducts. An embodiment of the subject device related to this invention operates at high temperatures and can withstand harsh chemical environments. Embodiments of the device are made on a single substrate. The devices can also be made on individual substrates and monitored individually as if they were part of an array on a single substrate. The device can incorporate sensing electrodes in the same environment, which allows the electrodes to be coplanar and, thus, keep manufacturing costs low. Embodiments of the device can provide improvements to sensitivity, selectivity, and signal interference via surface temperature control.

Notch-modifying xylosyltransferase structures support an S Ni-like retaining mechanism

DOE PAGES

Yu, Hongjun; Li, Huilin; Takeuchi, Megumi; ...

2015-09-28

A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a largemore » conformational change upon binding to the active site, providing a structural basis for substrate specificity. As a result, our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.« less

Notch-modifying xylosyltransferase structures support an S Ni-like retaining mechanism

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

Yu, Hongjun; Li, Huilin; Takeuchi, Megumi

A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a largemore » conformational change upon binding to the active site, providing a structural basis for substrate specificity. As a result, our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.« less

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

PubMed

Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

2013-01-24

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

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

Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory

Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

Functional associations between support use and forelimb shape in strepsirrhines and their relevance to inferring locomotor behavior in early primates.

PubMed

Fabre, Anne-Claire; Marigó, Judit; Granatosky, Michael C; Schmitt, Daniel

2017-07-01

The evolution of primates is intimately linked to their initial invasion of an arboreal environment. However, moving and foraging in this milieu creates significant mechanical challenges related to the presence of substrates differing in their size and orientation. It is widely assumed that primates are behaviorally and anatomically adapted to movement on specific substrates, but few explicit tests of this relationship in an evolutionary context have been conducted. Without direct tests of form-function relationships in living primates it is impossible to reliably infer behavior in fossil taxa. In this study, we test a hypothesis of co-variation between forelimb morphology and the type of substrates used by strepsirrhines. If associations between anatomy and substrate use exist, these can then be applied to better understand limb anatomy of extinct primates. The co-variation between each forelimb long bone and the type of substrate used was studied in a phylogenetic context. Our results show that despite the presence of significant phylogenetic signal for each long bone of the forelimb, clear support use associations are present. A strong co-variation was found between the type of substrate used and the shape of the radius, with and without taking phylogeny into account, whereas co-variation was significant for the ulna only when taking phylogeny into account. Species that use a thin branch milieu show radii that are gracile and straight and have a distal articular shape that allows for a wide range of movements. In contrast, extant species that commonly use large supports show a relatively robust and curved radius with an increased surface area available for forearm and hand muscles in pronated posture. These results, especially for the radius, support the idea that strepsirrhine primates exhibit specific skeletal adaptations associated with the supports that they habitually move on. With these robust associations in hand it will be possible to explore the same variables in extinct early primates and primate relatives and thus improve the reliability of inferences concerning substrate use in early primates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of Production Version of the NASA Improved Inorganic-Organic Separator

NASA Technical Reports Server (NTRS)

Sheibley, D.

1983-01-01

The technology of an inorganic-organic (I/O) separator, which demonstrated improved flexibility, reduced cost, production feasibility and improved cycle life was developed. Substrates to replace asbestos and waterbased separator coatings to replace the solvent based coatings were investigated. An improved fuel cell grade asbestos sheet was developed and a large scale production capability for the solvent based I/O separator was demonstrated. A cellulose based substrate and a nonwoven polypropylene fiber substrate were evaluated as replacements for the asbestos. Both the cellulose and polypropylene substrates were coated with solvent based and water based coatings to produce a modified I/O separator. The solvent based coatings were modified to produce aqueous separator coatings with acceptable separator properties. A single ply fuel cell grade asbestos with a binder (BTA) was produced. It has shown to be an acceptable substrate for the solvent and water based separator coatings, an acceptable absorber for alkaline cells, and an acceptable matrix for alkaline fuel cells. The original solvent based separator (K19W1), using asbestos as a substrate, was prepared.

Chemical pretreatment of lignocellulosic agroindustrial waste for methane production.

PubMed

Pellera, Frantseska-Maria; Gidarakos, Evangelos

2018-01-01

This study investigates the effect of different chemical pretreatments on the solubilization and the degradability of different solid agroindustrial waste, namely winery waste, cotton gin waste, olive pomace and juice industry waste. Eight different reagents were investigated, i.e. sodium hydroxide (NaOH), sodium bicarbonate (NaHCO 3 ), sodium chloride (NaCl), citric acid (H 3 Cit), acetic acid (AcOH), hydrogen peroxide (H 2 O 2 ), acetone (Me 2 CO) and ethanol (EtOH), under three condition sets resulting in treatments of varying intensity, depending on process duration, reagent dosage and temperature. Results indicated that chemical pretreatment under more severe conditions is more effective on the solubilization of lignocellulosic substrates, such as those of the present study and among the investigated reagents, H 3 Cit, H 2 O 2 and EtOH appeared to be the most effective to this regard. At the same time, although chemical pretreatment in general did not improve the methane potential of the substrates, moderate to high severity conditions were found to generally be the most satisfactory in terms of methane production from pretreated materials. In fact, moderate severity treatments using EtOH for winery waste, H 3 Cit for olive pomace and H 2 O 2 for juice industry waste and a high severity treatment with EtOH for cotton gin waste, resulted in maximum specific methane yield values. Ultimately, the impact of pretreatment parameters on the different substrates seems to be dependent on their characteristics, in combination with the specific mode of action of each reagent. The overall energy balance of such a system could probably be improved by using lower operating powers and higher solid to liquid ratios. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improved Advanced Actuated Hybrid Mirrors Final Report CRADA No. TC02130.0

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

Barbee, T. W.; Ealey, M. A.

2017-08-25

This was a collaborative effort to develop and demonstrate an improved Advanced Actuated Hybrid Mirrors (AAHM) for commercial or Government purposes. The AAHM consists of a nanolaminate film replicating a precision optical surface bonded to a Silicon Carbide (SiC) substrate with active figure control capability. The goal of this project was to further the development of specific AAHM technologies. The intent of the CRADA was to combine the expertise of LLNL and NG Xinetics in the manufacture and test of a very high quality AAHM, incorporating lessons learned from earlier joint efforts.

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

Apparatus and process for depositing hard coating in a nozzle orifice

DOEpatents

Flynn, P.L.; Giammarise, A.W.

1994-12-20

The present invention is directed to a process for coating the interior surfaces of an orifice in a substrate that forms a slurry fuel injection nozzle. In a specific embodiment, the nozzle is part of a fuel injection system for metering a coal-water slurry into a large, medium-speed, multi-cylinder diesel engine. In order to retard erosion of the orifice, the substrate is placed in a chemical vapor deposition (CVD) reaction chamber. A reaction gas is passed into the chamber at a gas temperature below its reaction temperature and is directed through the orifice in the substrate. The gas reaction temperature is a temperature at and above which the reaction gas deposits as a coating, and the reaction gas is of a composition whereby improved resistance to erosion by flow of the particulates in the slurry fuel is imparted by the deposited coating. Only the portion of the substrate in proximity to the orifice to be coated is selectively heated to at least the gas reaction temperature for effecting coating of the orifice's interior surfaces by the vapor deposited coating formed from the reaction gas. 2 figures.

Cellular response of preosteoblasts to nanograined/ultrafine-grained structures.

PubMed

Misra, R D K; Thein-Han, W W; Pesacreta, T C; Hasenstein, K H; Somani, M C; Karjalainen, L P

2009-06-01

Metallic materials with submicron- to nanometer-sized grains provide surfaces that are different from conventional polycrystalline materials because of the large proportion of grain boundaries with high free energy. In the study described here, the combination of cellular and molecular biology, materials science and engineering advances our understanding of cell-substrate interactions, especially the cellular activity between preosteoblasts and nanostructured metallic surfaces. Experiments on the effect of nano-/ultrafine grains have shown that cell attachment, proliferation, viability, morphology and spread are favorably modulated and significantly different from conventional coarse-grained structures. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on nanograined/ultrafine-grained substrate. These observations suggest enhanced cell-substrate interaction and activity. The differences in the cellular response on nanograined/ultrafine-grained and coarse-grained substrates are attributed to grain size and degree of hydrophilicity. The outcomes of the study are expected to reduce challenges to engineer bulk nanostructured materials with specific physical and surface properties for medical devices with improved cellular attachment and response. The data lay the foundation for a new branch of nanostructured materials for biomedical applications.

Method for improving the performance of oxidizable ceramic materials in oxidizing environments

NASA Technical Reports Server (NTRS)

Nagaraj, Bangalore A. (Inventor)

2002-01-01

Improved adhesion of thermal barrier coatings to nonmetallic substrates using a dense layer of ceramic on an underlying nonmetallic substrate that includes at least one oxidizable component. The improved adhesion occurs because the application of the dense ceramic layer forms a diffusion barrier for oxygen. This diffusion barrier prevents the oxidizable component of the substrate from decomposing. The present invention applies ceramic by a process that deposits a relatively thick and dense ceramic layer on the underlying substrate. The formation of the dense layer of ceramic avoids the problem of void formation associated with ceramic formation by most prior art thermal decomposition processes. The formation of voids has been associated with premature spalling of thermal barrier layers and other protective layers applied to substrates.

Two-dimensional modeling of water and heat fluxes in green roof substrates

NASA Astrophysics Data System (ADS)

Suarez, F. I.; Sandoval, V. P.

2016-12-01

Due to public concern towards sustainable development, greenhouse gas emissions and energy efficiency, green roofs have become popular in the last years. Green roofs integrate vegetation into infrastructures to reach additional benefits that minimize negative impacts of the urbanization. A properly designed green roof can reduce environmental pollution, noise levels, energetic requirements or surface runoff. The correct performance of green roofs depends on site-specific conditions and on each component of the roof. The substrate and the vegetation layers strongly influence water and heat fluxes on a green roof. The substrate is an artificial media that has an improved performance compared to natural soils as it provides critical resources for vegetation survival: water, nutrients, and a growing media. Hence, it is important to study the effects of substrate properties on green roof performance. The objective of this work is to investigate how the thermal and hydraulic properties affect the behavior of a green roof through numerical modeling. The substrates that were investigated are composed by: crushed bricks and organic soil (S1); peat with perlite (S2); crushed bricks (S3); mineral soil with tree leaves (S4); and a mixture of topsoil and mineral soil (S5). The numerical model utilizes summer-arid meteorological information to evaluate the performance of each substrate. Results show that the area below the water retention curve helps to define the substrate that retains more water. In addition, the non-linearity of the water retention curve can increment the water needed to irrigate the roof. The heat propagation through the roof depends strongly on the hydraulic behavior, meaning that a combination of a substrate with low thermal conductivity and more porosity can reduce the heat fluxes across the roof. Therefore, it can minimize the energy consumed of an air-conditioner system.

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

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

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

PubMed Central

Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio

2012-01-01

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182

Dissecting substrate specificities of the mitochondrial AFG3L2 protease.

PubMed

Ding, Bojian; Martin, Dwight W; Rampello, Anthony J; Glynn, Steven E

2018-06-22

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the pre-sequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degra-dation products from multiple substrates using mass spectrometry, we construct a peptidase specificity pro-file that displays constrained product lengths and is dominated by the identity of the residue at the P1' posi-tion, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, dis-criminating between potential substrates by recognizing accessible degron sequences, and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

PubMed Central

Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

2008-01-01

SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

PubMed Central

Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

2014-01-01

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

Nanoengineered Polystyrene Surfaces with Nanopore Array Pattern Alters Cytoskeleton Organization and Enhances Induction of Neural Differentiation of Human Adipose-Derived Stem Cells.

PubMed

Jung, Ae Ryang; Kim, Richard Y; Kim, Hyung Woo; Shrestha, Kshitiz Raj; Jeon, Seung Hwan; Cha, Kyoung Je; Park, Yong Hyun; Kim, Dong Sung; Lee, Ji Youl

2015-07-01

Human adipose-derived stem cells (hADSCs) can differentiate into various cell types depending on chemical and topographical cues. One topographical cue recently noted to be successful in inducing differentiation is the nanoengineered polystyrene surface containing nanopore array-patterned substrate (NP substrate), which is designed to mimic the nanoscale topographical features of the extracellular matrix. In this study, efficacies of NP and flat substrates in inducing neural differentiation of hADSCs were examined by comparing their substrate-cell adhesion rates, filopodia growth, nuclei elongation, and expression of neural-specific markers. The polystyrene nano Petri dishes containing NP substrates were fabricated by a nano injection molding process using a nickel electroformed nano-mold insert (Diameter: 200 nm. Depth of pore: 500 nm. Center-to-center distance: 500 nm). Cytoskeleton and filopodia structures were observed by scanning electron microscopy and F-actin staining, while cell adhesion was tested by vinculin staining after 24 and 48 h of seeding. Expression of neural specific markers was examined by real-time quantitative polymerase chain reaction and immunocytochemistry. Results showed that NP substrates lead to greater substrate-cell adhesion, filopodia growth, nuclei elongation, and expression of neural specific markers compared to flat substrates. These results not only show the advantages of NP substrates, but they also suggest that further study into cell-substrate interactions may yield great benefits for biomaterial engineering.

Microelectrophoretic apparatus and process

NASA Technical Reports Server (NTRS)

Grunbaum, B. W. (Inventor)

1978-01-01

New gel tray and lid assemblies designed for use in conjunction with slotted electrophoretic membranes were developed to take advantage of recently improved microelectrophoretic accessories which include a multisample applicator capable of applying up to 10 samples consecutively or simultaneously, and a temperature control plate for dissipating the heat produced by electrophoresis in a gel. The trays and membranes can be marketed ready for use as electrophoretic media or impregnated with various specific substrates and dyes

Improved measurement of extracellular enzymatic activities in subsurface sediments using competitive desorption treatment

NASA Astrophysics Data System (ADS)

Hoarfrost, Adrienne; Snider, Rachel; Arnosti, Carol

2017-02-01

Extracellular enzymatic activities initiate microbially-driven heterotrophic carbon cycling in subsurface sediments. While measurement of hydrolytic activities in sediments is fundamental to our understanding of carbon cycling, these measurements are often technically difficult due to sorption of organic substrates to the sediment matrix. Most methods that measure hydrolysis of organic substrates in sediments rely on recovery of a fluorophore or fluorescently-labeled target substrate from a sediment incubation. The tendency for substrates to sorb to sediments results in lower recovery of an added substrate, and can result in data that are unusable or difficult to interpret. We developed a treatment using competitive desorption of a fluorescently-labeled, high molecular weight organic substrate that improves recovery of the labeled substrate from sediment subsamples. Competitive desorption treatment improved recovery of the fluorescent substrate by a median of 66%, expanded the range of sediments for which activity measurements could be made, and was effective in sediments from a broad range of geochemical contexts. More reliable measurements of hydrolytic activities in sediments will yield usable and more easily interpretable data from a wider range of sedimentary environments, enabling better understanding of microbially-catalyzed carbon cycling in subsurface environments.

Visual cues for woodpeckers: light reflectance of decayed wood varies by decay fungus

USGS Publications Warehouse

O'Daniels, Sean T.; Kesler, Dylan C.; Mihail, Jeanne D.; Webb, Elisabeth B.; Werner, Scott J.

2018-01-01

The appearance of wood substrates is likely relevant to bird species with life histories that require regular interactions with wood for food and shelter. Woodpeckers detect decayed wood for cavity placement or foraging, and some species may be capable of detecting trees decayed by specific fungi; however, a mechanism allowing for such specificity remains unidentified. We hypothesized that decay fungi associated with woodpecker cavity sites alter the substrate reflectance in a species-specific manner that is visually discriminable by woodpeckers. We grew 10 species of wood decay fungi from pure cultures on sterile wood substrates of 3 tree species. We then measured the relative reflectance spectra of decayed and control wood wafers and compared them using the receptor noise-limited (RNL) color discrimination model. The RNL model has been used in studies of feather coloration, egg shells, flowers, and fruit to model how the colors of objects appear to birds. Our analyses indicated 6 of 10 decayed substrate/control comparisons were above the threshold of discrimination (i.e., indicating differences discriminable by avian viewers), and 12 of 13 decayed substrate comparisons were also above threshold for a hypothetical woodpecker. We conclude that woodpeckers should be capable of visually detecting decayed wood on trees where bark is absent, and they should also be able to detect visually species-specific differences in wood substrates decayed by fungi used in this study. Our results provide evidence for a visual mechanism by which woodpeckers could identify and select substrates decayed by specific fungi, which has implications for understanding ecologically important woodpecker–fungus interactions.

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.

PubMed

Wecksler, Aaron T; Garcia, Natalie K; Holman, Theodore R

2009-09-15

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

PubMed Central

Campbell, T B; Cech, T R

1995-01-01

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519

Efficient production of l-lactic acid by an engineered Thermoanaerobacterium aotearoense with broad substrate specificity

PubMed Central

2013-01-01

Background Efficient conversion of lignocellulosic biomass to optically pure lactic acid is a key challenge for the economical production of biodegradable poly-lactic acid. A recently isolated strain, Thermoanaerobacterium aotearoense SCUT27, is promising as an efficient lactic acid production bacterium from biomass due to its broad substrate specificity. Additionally, its strictly anaerobic and thermophilic characteristics suppress contamination from other microoragnisms. Herein, we report the significant improvements of concentration and yield in lactic acid production from various lignocellulosic derived sugars, achieved by the carbon flux redirection through homologous recombination in T. aotearoense SCUT27. Results T. aotearoense SCUT27 was engineered to block the acetic acid formation pathway to improve the lactic acid production. The genetic manipulation resulted in 1.8 and 2.1 fold increase of the lactic acid yield using 10 g/L of glucose or 10 g/L of xylose as substrate, respectively. The maximum l-lactic acid yield of 0.93 g/g glucose with an optical purity of 99.3% was obtained by the engineered strain, designated as LA1002, from 50 g/L of substrate, which is very close to the theoretical value (1.0 g/g of glucose). In particular, LA1002 produced lactic acid at an unprecedented concentration up to 3.20 g/L using 10 g/L xylan as the single substrate without any pretreatment after 48 h fermentation. The non-sterilized fermentative production of l-lactic acid was also carried out, achieving values of 44.89 g/L and 0.89 g/g mixed sugar for lactic acid concentration and yield, respectively. Conclusions Blocking acetic acid formation pathway in T. aotearoense SCUT27 increased l-lactic acid production and yield dramatically. To our best knowledge, this is the best performance of fermentation on lactic acid production using xylan as the sole carbon source, considering the final concentration, yield and fermentation time. In addition, it should be mentioned that the performance of non-sterilized simultaneous fermentation from glucose and xylose was very close to that of normal sterilized cultivation. All these results used the mutant strain, LA1002, indicated that it is a new promising candidate for the effective production of optically pure l-lactic acid from lignocellulosic biomass. PMID:23985133

Integrated cellulosic enzymes hydrolysis and fermentative advanced yeast bioconversion solution ready for biomass biorefineries

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

Kumar, Manoj

2011-05-04

These are slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme,more » as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Thermostable Cellulases: Why & How?

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

Kumar, Manoj

2010-03-24

These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

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

Kumar, Manoj

2011-02-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Highly Efficient Thermostable DSM Cellulases: Why & How?

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

Kumar, Manoj

2011-04-26

These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase componentmore » enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Fully Integrated Lignocellulosic Biorefinery with Onsite Production of Enzymes and Yeast

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

Kumar, Manoj

2010-06-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Radio frequency diodes and circuits fabricated via adhesion lithography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Georgiadou, Dimitra G.; Semple, James; Wyatt-Moon, Gwenhivir; Anthopoulos, Thomas D.

2016-09-01

The commercial interest in Radio Frequency Identification (RFID) tags keeps growing, as new application sectors, spanning from healthcare to electronic article surveillance (EAS) and personal identification, are constantly emerging for these types of electronic devices. The increasing demand for the so-called "smart labels" necessitates their high throughput manufacturing, and indeed on thin flexible substrates, that will reduce the cost and render them competitive to the currently widely employed barcodes. Adhesion Lithography (a-Lith) is a novel patterning technique that allows the facile high yield fabrication of co-planar large aspect ratio (<100,000) metal electrodes separated by a sub-20 nm gap on large area substrates of any type. Deposition of high mobility semiconductors from their solution at low, compatible with plastic substrates, temperatures and application of specific processing protocols can dramatically improve the performance of the fabricated Schottky diodes. It will be shown that in this manner both organic and inorganic high speed diodes and rectifiers can be obtained, operating at frequencies much higher than the 13.56 MHz benchmark, currently employed in passive RFID tags and near filed communications (NFC). This showcases the universality of this method towards fabricating high speed p- and n-type diodes, irrespective of the substrate, simply based on the extreme downscaling of key device dimensions obtained in these nanoscale structures. The potential for scaling up this technique at low cost, combined with the significant performance optimisation and improved functionality that can be attained through intelligent material selection, render a-Lith unique within the field of plastic electronics.

Photovoltaic options for solar electric propulsion

NASA Technical Reports Server (NTRS)

Stella, Paul M.; Flood, Dennis J.

1990-01-01

During the past decade, a number of advances have occurred in solar cell and array technology. These advances have lead to performance improvement for both conventional space arrays and for advanced technology arrays. Performance enhancements have occurred in power density, specific power, and environmental capability. Both state-of-the-art and advanced development cells and array technology are discussed. Present technology will include rigid, rollout, and foldout flexible substrate designs, with silicon and GaAs solar cells. The use of concentrator array systems is also discussed based on both DOD and NASA efforts. The benefits of advanced lightweight array technology, for both near term and far term utilization, and of advanced high efficiency, thin, radiation resistant cells is examined. This includes gallium arsenide on germaniun substrates, indium phosphide, and thin film devices such as copper indium diselenide.

Asymmetry of the velocity-matching steps in YBCO long Josephson junctions

NASA Astrophysics Data System (ADS)

Revin, L. S.; Pankratov, A. L.; Chiginev, A. V.; Masterov, D. V.; Parafin, A. E.; Pavlov, S. A.

2018-04-01

We carry out experimental and theoretical investigations into the effect of the vortex chain propagation on the current-voltage characteristics of YBa2Cu3O7-δ (YBCO) long Josephson junctions. Samples of YBCO Josephson junctions, fabricated on 24° [001]-tilt bicrystal substrates, have been measured. The improved technology has allowed us to observe and study the asymmetry of the current-voltage characteristics with opposite magnetic fields (Revin et al 2012 J. Appl. Phys. 114 243903), which we believe occurs due to anisotropy of bicrystal substrates (Kupriyanov et al (2013 JETP Lett. 95 289)). Specifically, we examine the flux-flow resonant steps versus the external magnetic field, and study the differential resistance and its relation to oscillation power for opposite directions of vortex propagation.

Effect of oxidation of the non-catalytic β-propeller domain on the substrate specificity of prolyl oligopeptidase from Pleurotus eryngii.

PubMed

Tokai, Shota; Bito, Tomohiro; Shimizu, Katsuhiko; Arima, Jiro

2017-05-27

Enzymes belonging to the S9 family of prolyl oligopeptidases are of interest because of their pharmacological importance and have a non-catalytic β-propeller domain. In this study, we found that the oxidation of Met203, which lies on surface of the β-propeller domain, leads to change in the substrate specificity of eryngase, an enzyme from Pleurotus eryngii and a member of the S9 family of prolyl oligopeptidases. The activity of eryngase for L-Phe-p-nitroanilide was maintained following hydrogen peroxide treatment but was dramatically reduced for other p-nitroanilide substrates. MALDI-TOF MS analysis using tryptic peptides of eryngase indicated that the change in substrate specificity was triggered by oxidizing Met203 to methionine sulfoxide. In addition, mutations of Met203 to smaller residues provided specificities similar to those observed following oxidation of the wild-type enzyme. Substitution of Met203 with Phe significantly decreased activity, indicating that Met203 may be involved in substrate gating. Copyright © 2017 Elsevier Inc. All rights reserved.

Latent fingermark development on a range of porous substrates using ninhydrin analogs--a comparison with ninhydrin and 1,8-diazofluoren.

PubMed

Berdejo, Stephanie; Rowe, Mark; Bond, John W

2012-03-01

Three relatively new reagents for developing latent fingermarks on porous substrates, 1,2-indandione (IND), 5-methylthioninhydrin (5-MTN), and lawsone, are compared with the more widely used ninhydrin and 1,8-diazofluoren (DFO). Developed latent fingermark visualization on 10 different substrates comprising colored papers, cardboard, and cellophane rather than conventional printer and writing/notepad paper is assessed using latent fingermark deposits from 48 donors. Results show improved fluorescent fingermark visualization using IND compared with DFO on a range of colored cardboards and thick white paper, thus extending the range of substrates known to yield improved visualization with IND. Adding zinc chloride to IND failed to yield any further improvement in fluorescent fingermark visualization. 5-MTN (with and without zinc chloride posttreatment) showed no improvement in visualization compared with ninhydrin and DFO although visible fingermarks were developed. Lawsone produced fluorescent visible fingermarks only with white substrates, which were inferior to those produced with DFO. © 2011 American Academy of Forensic Sciences.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

Chronic administration of DSP-7238, a novel, potent, specific and substrate-selective DPP IV inhibitor, improves glycaemic control and beta-cell damage in diabetic mice.

PubMed

Furuta, Y; Horiguchi, M; Sugaru, E; Ono-Kishino, M; Otani, M; Sakai, M; Masui, Y; Tsuchida, A; Sato, Y; Takubo, K; Hochigai, H; Kimura, H; Nakahira, H; Nakagawa, T; Taiji, M

2010-05-01

The purpose of this study is to assess the in vitro enzyme inhibition profile of DSP-7238, a novel non-cyanopyrrolidine dipeptidyl peptidase (DPP) IV inhibitor and to evaluate the acute and chronic effects of this compound on glucose metabolism in two different mouse models of type 2 diabetes. The in vitro enzyme inhibition profile of DSP-7238 was assessed using plasma and recombinant enzymes including DPP IV, DPP II, DPP8, DPP9 and fibroblast activation protein alpha (FAPalpha) with fluorogenic substrates. The inhibition type was evaluated based on the Lineweaver-Burk plot. Substrate selectivity of DSP-7238 and comparator DPP IV inhibitors (vildagliptin, sitagliptin, saxagliptin and linagliptin) was evaluated by mass spectrometry based on the changes in molecular weight of peptide substrates caused by release of N-terminal dipeptides. In the in vivo experiments, high-fat diet-induced obese (DIO) mice were subjected to oral glucose tolerance test (OGTT) following a single oral administration of DSP-7238. To assess the chronic effects of DSP-7238 on glycaemic control and pancreatic beta-cell damage, DSP-7238 was administered for 11 weeks to mice made diabetic by a combination of high-fat diet (HFD) and a low-dose of streptozotocin (STZ). After the dosing period, HbA1c was measured and pancreatic damage was evaluated by biological and histological analyses. DSP-7238 and sitagliptin both competitively inhibited recombinant human DPP IV (rhDPP IV) with K(i) values of 0.60 and 2.1 nM respectively. Neither vildagliptin nor saxagliptin exhibited competitive inhibition of rhDPP IV. DSP-7238 did not inhibit DPP IV-related enzymes including DPP8, DPP9, DPP II and FAPalpha, whereas vildagliptin and saxagliptin showed inhibition of DPP8 and DPP9. Inhibition of glucagon-like peptide-1 (GLP-1) degradation by DSP-7238 was apparently more potent than its inhibition of chemokine (C-X-C motif) ligand 10 (IP-10) or chemokine (C-X-C motif) ligand 12 (SDF-1alpha) degradation. In contrast, vildagliptin and saxagliptin showed similar degree of inhibition of degradation for all the substrates tested. Compared to treatment with the vehicle, single oral administration of DSP-7238 dose-dependently decreased plasma DPP IV activity and improved glucose tolerance in DIO mice. In addition, DSP-7238 significantly decreased HbA1c and ameliorated pancreatic damage following 11 weeks of chronic treatment in HFD/STZ mice. We have shown in this study that DSP-7238 is a potent DPP IV inhibitor that has high specificity for DPP IV and substrate selectivity against GLP-1. We have also found that chronic treatment with DSP-7238 improves glycaemic control and ameliorates beta-cell damage in a mouse model with impaired insulin sensitivity and secretion. These findings indicate that DSP-7238 may be a new therapeutic agent for the treatment of type 2 diabetes.

X-Ray Fluorescence Determination of Sulphur in Oils by a Thin Film Method.

DTIC Science & Technology

1983-09-01

thickness. The procedure utilises a mixture of samp and an alkyd resin to improve adhesion to a Mylar substrate and to reduce sample flow during...thickness. The procedure utilises a mixture of sample and an alkyd resin to improve adhesion to a Mylar substrate and to reduce sample flow during...film. By incorporating an alkyd resin into the sample mixture the adhesion of the oil film to the Mylar sheet substrate is improved to the extent that

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

PubMed

Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

PubMed Central

Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion. PMID:24066121

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

PubMed

Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

2015-11-17

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

PubMed

Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J

2018-02-28

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

[Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

PubMed

Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

2014-03-01

In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

PubMed

Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

2014-04-01

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

Innovative nanostructures for highly sensitive vibrational biosensing (Conference Presentation)

NASA Astrophysics Data System (ADS)

Popp, Juergen; Mayerhöfer, Thomas; Cialla-May, Dana; Weber, Karina; Huebner, Uwe

2016-03-01

Employing vibrational spectroscopy (IR-absorption and Raman spectroscopy) allows for the labelfree detection of molecular specific fingerprints of inorganic, organic and biological substances. The sensitivity of vibrational spectroscopy can be improved by several orders of magnitude via the application of plasmonic active surfaces. Within this contribution we will discuss two such approaches, namely surface enhanced Raman spectroscopy (SERS) as well as surface enhanced IR absorption (SEIRA). It will be shown that SERS using metal colloids as SERS active substrate in combination with a microfluidic lab-on-a-chip (LOC) device enables high throughput and reproducible measurements with highest sensitivity and specificity. The application of such a LOC-SERS approach for therapeutic drug monitoring (e.g. quantitative detection of antibiotics in a urine matrix) will be presented. Furthermore, we will introduce innovative bottom-up strategies to prepare SERS-active nanostructures coated with a lipophilic sensor layer as one-time use SERS substrates for specific food analysis (e.g. quantitative detection of toxic food colorants). The second part of this contribution presents a slit array metamaterial perfect absorber for IR sensing applications consisting of a dielectric layer sandwiched between two metallic layers of which the upper layer is perforated with a periodic array of slits. Light-matter interaction is greatly amplified in the slits, where also the analyte is concentrated, as the surface of the substrate is covered by a thin silica layer. Thus, already small concentrations of analytes down to a monolayer can be detected by refractive index sensing and identified by their spectral fingerprints with a standard mid-infrared lab spectrometer.

A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

PubMed

Stewart, James J P

2016-11-01

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

Lava-substrate heat transfer: Laboratory experiments and thermodynamic modeling

NASA Astrophysics Data System (ADS)

Rumpf, M.; Fagents, S. A.; Hamilton, C. W.; Wright, R.; Crawford, I.

2012-12-01

We have performed laboratory experiments and numerical modeling to investigate the heat transfer from a lava flow into various substrate materials, focusing on the effects of the differing thermophysical properties of substrate materials. Initial motivation for this project developed from the desire to understand the loss of solar wind volatiles embedded in lunar regolith deposits that were subsequently covered by a lava flow. The Moon lacks a significant atmosphere and magnetosphere, leaving the surface regolith exposed to bombardment by solar flare and solar wind particles, and by the cosmogenic products of galactic cosmic rays. Preservation of particle-rich regolith deposits may have occurred by the emplacement of an active lava flow on top of the regolith layer, provided the embedded particles survive heating by the lava. During future expeditions to the lunar surface, ancient regolith deposits could be sampled through surface drilling to extract the extra-lunar particles, revealing a history of the solar activity and galactic events not available on the Earth. This project also has important implications for terrestrial lava flows, particularly in the prediction of lava flow hazards. Lava erupted on Earth may be emplaced on various substrates, including solid lava rock, volcanic tephra, sands, soils, etc. The composition, grain size, consolidation, moisture content, etc. of these materials will vary greatly and have different effects on the cooling of the flow. Accounting for specific properties of the substrate could be an important improvement in lava flow models We have performed laboratory experiments in collaboration with the Department of Art and Art History at the University of Hawaii at Manoa in which ~5-6 kg of basalt, collected at Kilauea Volcano, Hawaii, is melted to ~1200 °C. The lava is poured into a device constructed of calcium silicate sheeting that has been filled with a solid or particulate substrate material and embedded with thermocouples. Internal temperatures are monitored by the thermocouple array, while external temperatures are monitored by a Forward Looking Infrared Radiometer (FLIR) video camera. The experimental data thus describe the cooling rates of the system, and reveal the release of latent heat of crystallization within the cooling lava. These experiments have been conducted in conjunction with numerical simulations of the heat transfer from a lava flow into various substrates, to quantify the depth reached by the heat pulse as it penetrates the substrate. Models include material-specific, temperature-dependent thermophysical properties, including thermal conductivity, specific heat capacity, and latent heat of crystallization. We find that particulate materials, such as lunar regolith, sand, and soils will be heated to depths shallower than solid materials. In addition, the particulate materials will act as insulators, shielding the lava flow from basal cooling and maintaining high temperatures in the flow core. These results suggest that lava flows emplaced on a dry particulate terrain will remain above solidus for a longer duration, allowing the lava to flow further than when emplaced on a solid substrate.

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways

PubMed Central

Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

2014-01-01

Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively. PMID:24847354

Synthesis and property of spinel porous ZnMn2O4 microspheres

NASA Astrophysics Data System (ADS)

Guo, N.; Wei, X. Q.; Deng, X. L.; Xu, X. J.

2015-11-01

Mesoporous ternary zinc manganese oxides on the Ti sheet substrate are prepared by easy and fast hydrothermal method for the first time. The obtained ZnMn2O4 materials with homogenously distributed pores have been characterized by XRD, SEM and Raman spectra, which show the good crystal phase and particles for improving supercapacitive performance. XRD and SEM images show that the as-prepared samples have good crystallinity, and ZnMn2O4 microsphere has an average diameter of 10 μm. In addition, ZnMn2O4 are also characterized in 2 M KOH solution using three-electrode system. In the work, we study that different substrates (Ti, carbon and nickel foam) have an important effect on the electrochemical performance of the samples. The research of cyclic voltammogram (CV) indicates that the obtained specific capacitance (155 F g-1) values on nickel foam substrate for the ZnMn2O4 microspheres are higher than the values reported for some inexpensive oxides. However, the specific capacitance of all ZnMn2O4 samples has almost no change at two different scan rates which shows good long-term cycling stability. The electrochemical impedance spectroscopy with a small resistance reveals that the as-synthesized samples have good frequency response characteristics. These results indicate that the unique ZnMn2O4 electrode would be a promising electrode for high-performance supercapacitor applications.

Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

ERIC Educational Resources Information Center

Linenberger, Kimberly J.; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Wear and Adhesive Failure of Al2O3 Powder Coating Sprayed onto AISI H13 Tool Steel Substrate

NASA Astrophysics Data System (ADS)

Amanov, Auezhan; Pyun, Young-Sik

2016-07-01

In this study, an alumina (Al2O3) ceramic powder was sprayed onto an AISI H13 hot-work tool steel substrate that was subjected to sanding and ultrasonic nanocrystalline surface modification (UNSM) treatment processes. The significance of the UNSM technique on the adhesive failure of the Al2O3 coating and on the hardness of the substrate was investigated. The adhesive failure of the coating sprayed onto sanded and UNSM-treated substrates was investigated by a micro-scratch tester at an incremental load. It was found, based on the obtained results, that the coating sprayed onto the UNSM-treated substrate exhibited a better resistance to adhesive failure in comparison with that of the coating sprayed onto the sanded substrate. Dry friction and wear property of the coatings sprayed onto the sanded and UNSM-treated substrates were assessed by means of a ball-on-disk tribometer against an AISI 52100 steel ball. It was demonstrated that the UNSM technique controllably improved the adhesive failure of the Al2O3 coating, where the critical load was improved by about 31%. Thus, it is expected that the application of the UNSM technique to an AISI H13 tool steel substrate prior to coating may delay the adhesive failure and improve the sticking between the coating and the substrate thanks to the modified and hardened surface.

An assessment of catalytic residue 3D ensembles for the prediction of enzyme function.

PubMed

Žváček, Clemens; Friedrichs, Gerald; Heizinger, Leonhard; Merkl, Rainer

2015-11-04

The central element of each enzyme is the catalytic site, which commonly catalyzes a single biochemical reaction with high specificity. It was unclear to us how often sites that catalyze the same or highly similar reactions evolved on different, i. e. non-homologous protein folds and how similar their 3D poses are. Both similarities are key criteria for assessing the usability of pose comparison for function prediction. We have analyzed the SCOP database on the superfamily level in order to estimate the number of non-homologous enzymes possessing the same function according to their EC number. 89% of the 873 substrate-specific functions (four digit EC number) assigned to mono-functional, single-domain enzymes were only found in one superfamily. For a reaction-specific grouping (three digit EC number), this value dropped to 35%, indicating that in approximately 65% of all enzymes the same function evolved in two or more non-homologous proteins. For these isofunctional enzymes, structural similarity of the catalytic sites may help to predict function, because neither high sequence similarity nor identical folds are required for a comparison. To assess the specificity of catalytic 3D poses, we compiled the redundancy-free set ENZ_SITES, which comprises 695 sites, whose composition and function are well-defined. We compared their poses with the help of the program Superpose3D and determined classification performance. If the sites were from different superfamilies, the number of true and false positive predictions was similarly high, both for a coarse and a detailed grouping of enzyme function. Moreover, classification performance did not improve drastically, if we additionally used homologous sites to predict function. For a large number of enzymatic functions, dissimilar sites evolved that catalyze the same reaction and it is the individual substrate that determines the arrangement of the catalytic site and its local environment. These substrate-specific requirements turn the comparison of catalytic residues into a weak classifier for the prediction of enzyme function.

Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G).

PubMed

Han, Ruizhi; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian

2013-07-01

In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of L-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2% higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

PubMed

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Microstructure and Antiwear Property of Laser Cladding Ni-Co Duplex Coating on Copper.

PubMed

Wang, Yiyong; Liang, Zhipeng; Zhang, Junwei; Ning, Zhe; Jin, Hui

2016-07-28

Ni-Co duplex coatings were cladded onto Cu to improve the antiwear properties of Cu products. Prior to laser cladding, n-Al₂O₃/Ni layers were introduced as interlayers between laser cladding coatings and Cu substrates to improve the laser absorptivity of these substrates and ensure defect-free laser cladding coatings. The structure and morphology of the coatings were characterized by scanning electron microscopy and optical microscopy, and the phases of the coatings were analyzed by X-ray diffraction. Their hardness was measured using a microhardness tester. Experimental results showed that defect-free composite coatings were obtained and that the coatings were metallurgically bonded to the substrates. The surface of the Ni-Co duplex coatings comprised a Co-based solid solution, Cr₇C₃, (Fe,Ni) 23 C₆, and other strengthening phases. The microhardness and wear resistance of the duplex coatings were significantly improved compared with the Cu substrates. The average microhardness of the cladded coatings was 845.6 HV, which was approximately 8.2 times greater than that of the Cu substrates (102.6 HV). The volume loss of the Cu substrates was approximately 7.5 times greater than that of the Ni-Co duplex coatings after 60 min of sliding wear testing. The high hardness of and lack of defects in the Ni-Co duplex coatings reduced the plastic deformation and adhesive wear of the Cu substrates, resulting in improved wear properties.

Photocatalytic activity of titania coatings synthesised by a combined laser/sol–gel technique

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

Adraider, Y.; Pang, Y.X., E-mail: F6098038@tees.ac.uk; Nabhani, F.

2014-06-01

Highlights: • Sol–gel method was used to prepare titania coatings. • Titania thin films were coated on substrate surface by dip coating. • Fibre laser was employed to irradiate the titania coated surfaces. • Photocatalytic efficiency of titania coatings was significantly improved after laser processing. - Abstract: Titania coatings were prepared using sol–gel method and then applied on the substrate surface by dip coating. Fibre laser (λ = 1064 nm) in continuous wave mode was used to irradiate the titania coated surfaces at different specific energies. The ATR-FTIR, XRD, SEM, EDS and contact angle measurement were employed to analyse surfacemore » morphology, phase composition and crystalline structure of laser-irradiated titania coatings, whilst the photocatalytic activity was evaluated by measuring the decomposition of methylene blue (MB) after exposure to the visible light for various illumination times. Results showed that the laser-irradiated titania coatings demonstrate significant different composition and microstructure in comparison with the as-coated from the same sol–gel titania. Photocatalytic efficiency of titania coatings was significantly improved after laser processing. The photocatalytic activity of laser-irradiated titania coatings was higher than that of the as-coated titania. The titania coating processed at laser specific energy of 6.5 J/mm{sup 2} exhibited the highest photocatalytic activity among all titania samples.« less

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization

PubMed Central

Kim, Tae-Su; Patel, Sanjay K. S.; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-01-01

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s−1 toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP+ (vs. only 2.5% relative activity with NAD+). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP+-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol. PMID:27633501

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization.

PubMed

Kim, Tae-Su; Patel, Sanjay K S; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-09-16

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents.

PubMed

Tsai, Shau-Wei; Chen, Chun-Chi; Yang, Hung-Shien; Ng, I-Son; Chen, Teh-Liang

2006-08-01

In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.

Ultra-low roughness magneto-rheological finishing for EUV mask substrates

NASA Astrophysics Data System (ADS)

Dumas, Paul; Jenkins, Richard; McFee, Chuck; Kadaksham, Arun J.; Balachandran, Dave K.; Teki, Ranganath

2013-09-01

EUV mask substrates, made of titania-doped fused silica, ideally require sub-Angstrom surface roughness, sub-30 nm flatness, and no bumps/pits larger than 1 nm in height/depth. To achieve the above specifications, substrates must undergo iterative global and local polishing processes. Magnetorheological finishing (MRF) is a local polishing technique which can accurately and deterministically correct substrate figure, but typically results in a higher surface roughness than the current requirements for EUV substrates. We describe a new super-fine MRF® polishing fluid whichis able to meet both flatness and roughness specifications for EUV mask blanks. This eases the burden on the subsequent global polishing process by decreasing the polishing time, and hence the defectivity and extent of figure distortion.

Bacterial growth and substrate degradation by BTX-oxidizing culture in response to salt stress.

PubMed

Lee, Chi-Yuan; Lin, Ching-Hsing

2006-01-01

Interactions between microbial growth and substrate degradation are important in determining the performance of trickle-bed bioreactors (TBB), especially when salt is added to reduce biomass formation in order to alleviate media clogging. This study was aimed at quantifying salinity effects on bacterial growth and substrate degradation, and at acquiring kinetic information in order to improve the design and operation of TBB. Experiment works began by cultivating a mixed culture in a chemostat reactor receiving artificial influent containing a mixture of benzene, toluene, and xylene (BTX), followed by using the enrichment culture to degrade the individual BTX substrates under a particular salinity, which ranged 0-50 g l(-1) in batch mode. Then, the measured concentrations of biomass and residual substrate versus time were analyzed with the microbial kinetics; moreover, the obtained microbial kinetic constants under various salinities were modeled using noncompetitive inhibition kinetics. For the three substrates the observed bacterial yields appeared to be decreased from 0.51-0.74 to 0.20-0.22 mg mg(-1) and the maximum specific rate of substrate utilization, q, declined from 0.25-0.42 to 0.07-0.11 h(-1), as the salinity increased from 0 to 50 NaCl g l(-1). The NaCl acted as noncompetitive inhibitor, where the modeling inhibitions of the coefficients, K ( T(S)), were 22.7-29.7 g l(-1) for substrate degradation and K ( T(mu)), 13.0-19.0 g l(-1), for biomass formation. The calculated ratios for the bacterial maintenance rate, m (S), to q, further indicated that the percentage energy spent on maintenance increased from 19-24 to 86-91% as salinity level increased from 0 to 50 g l(-1). These results revealed that the bacterial growth was more inhibited than substrate degradation by the BTX oxidizers under the tested salinity levels. The findings from this study demonstrate the potential of applying NaCl salt to control excessive biomass formation in biotrickling filters.

Effect of Alignment on Transport Properties of Carbon Nanotube/Metallic Junctions

NASA Technical Reports Server (NTRS)

Wincheski, Buzz; Namkung, Min; Smits, Jan; Williams, Phillip; Harvey, Robert

2003-01-01

Ballistic and spin coherent transport in single walled carbon nanotubes (SWCNT) are predicted to enable high sensitivity single-nanotube devices for strain and magnetic field sensing. Based upon these phenomena, electron beam lithography procedures have been developed to study the transport properties of purified HiPCO single walled carbon nanotubes for development into sensory materials for nondestructive evaluation. Purified nanotubes are dispersed in solvent suspension and then deposited on the device substrate before metallic contacts are defined and deposited through electron beam lithography. This procedure produces randomly dispersed ropes, typically 2 - 20 nm in diameter, of single walled carbon nanotubes. Transport and scanning probe microscopy studies have shown a good correlation between the junction resistance and tube density, alignment, and contact quality. In order to improve transport properties of the junctions a technique has been developed to align and concentrate nanotubes at specific locations on the substrate surface. Lithographic techniques are used to define local areas where high frequency electric fields are to be concentrated. Application of the fields while the substrate is exposed to nanotube-containing solution results in nanotube arrays aligned with the electric field lines. A second electron beam lithography layer is then used to deposit metallic contacts across the aligned tubes. Experimental measurements are presented showing the increased tube alignment and improvement in the transport properties of the junctions.

QM/MM Investigation of Substrate and Product Specificities of Suv4-20h2: How Does This Enzyme Generate Dimethylated H4K20 from Monomethylated Substrate?

PubMed

Qian, Ping; Guo, Haobo; Wang, Liang; Guo, Hong

2017-06-13

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on histone proteins in the regulation of chromatin structure and gene expression. In contrast to many other PKMTs for which unmodified lysine is the methylation target, the enzymes in the Suv4-20 family are able to generate dimethylated product (H4K20me2) based exclusively on the monomethylated H4K20 substrate (H4K20me1). The origin of such substrate/product specificity is still not clear. Here, molecular dynamics (MD) and free energy (potential of mean force) simulations are undertaken using quantum mechanical/molecular mechanical (QM/MM) potentials to understand the substrate/product specificities of Suv4-20h2, a member of the Suv4-20 family. The free energy barriers for mono-, di-, and trimethylation in Suv4-20h2 obtained from the simulations are found to be well correlated with the specificities observed experimentally with the allowed dimethylation based on the H4K20me1 substrate and prohibited monomethylation and trimethylation based on H4K20 and H4K20me2, respectively. It is demonstrated that the reason for the relatively efficient dimethylation is an effective transition state (TS) stabilization through strengthening the CH···O interactions as well as the presence of a cation-π interaction at the transition state. The simulations also show that the failures of Suv4-20h2 to catalyze monomethylation and trimethylation are due, respectively, to a less effective TS stabilization and inability of the reactant complex containing H4K20me2 to adopt a reactive (near attack) configuration for methyl transfer. The results suggest that care must be exercised in the prediction of the substrate specificity based only on the existence of near attack configurations in substrate complexes.

Active substrates improving sensitivity in biomedical fluorescence microscopy

NASA Astrophysics Data System (ADS)

Le Moal, E.; Leveque-Fort, S.; Fort, E.; Lacharme, J.-P.; Fontaine-Aupart, M.-P.; Ricolleau, C.

2005-08-01

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

Improvement in surface conditions of electroplated Fe-Pt thick-film magnets

NASA Astrophysics Data System (ADS)

Yanai, T.; Honda, J.; Hamamura, R.; Omagari, Y.; Yamada, H.; Fujita, N.; Takashima, K.; Nakano, M.; Fukunaga, H.

2018-05-01

Fe-Pt thick-films were electroplated on Ta, Ti, Co, Ni, and Cu plates (substrates) using a direct current, and the surface morphology, the magnetic properties, and the crystal structure of the films were evaluated. The films plated on the Co, Ni, and Cu substrates showed much smooth surface compared with those for the Ta and Ti ones, and we confirmed that the Cu plate was the most attractive substrate due to very small cracks after an annealing for L10 ordering. High coercivity (>800 kA/m) for the Cu substrate is almost the same as that for our previous study in which we employed the Ta substrate, and we found that the Cu plate is a hopeful substrate to improve the surface conditions of electroplated Fe-Pt thick-film magnets.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

PubMed

Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe

2016-06-08

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

Insights into the Specificity of Lysine Acetyltransferases

DOE PAGES

Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

2014-11-07

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

Wafer scale BN on sapphire substrates for improved graphene transport.

PubMed

Vangala, Shivashankar; Siegel, Gene; Prusnick, Timothy; Snure, Michael

2018-06-11

Wafer scale (2") BN grown by metal organic chemical vapor deposition (MOCVD) on sapphire was examined as a weakly interacting dielectric substrate for graphene, demonstrating improved transport properties over conventional sapphire and SiO 2 /Si substrates. Chemical vapor deposition grown graphene was transferred to BN/sapphire substrates for evaluation of more than 30 samples using Raman and Hall effects measurements. A more than 2x increase in Hall mobility and 10x reduction in sheet carrier density was measured for graphene on BN/sapphire compared to sapphire substrates. Through control of the MOCVD process, BN films with roughness ranging from <0.1 nm to >1 nm were grown and used to study the effects of substrate roughness on graphene transport. Arrays of graphene field effect transistors were fabricated on 2" BN/sapphire substrates demonstrating scalability and device performance enhancement.

The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans.

PubMed

Nisha, M; Satyanarayana, T

2015-07-01

In order to understand the role of N1 domain (1-257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir-Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability.

Quantum dot-based microfluidic biosensor for cancer detection

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

Ghrera, Aditya Sharma; School of Engineering and Technology, ITM University, Gurgaon-122017; Pandey, Chandra Mouli

2015-05-11

We report results of the studies relating to fabrication of an impedimetric microfluidic–based nucleic acid sensor for quantification of DNA sequences specific to chronic myelogenous leukemia (CML). The sensor chip is prepared by patterning an indium–tin–oxide (ITO) coated glass substrate via wet chemical etching method followed by sealing with polydimethylsiloxane (PDMS) microchannel for fluid control. The fabricated microfluidic chip comprising of a patterned ITO substrate is modified by depositing cadmium selenide quantum dots (QCdSe) via Langmuir–Blodgett technique. Further, the QCdSe surface has been functionalized with specific DNA probe for CML detection. The probe DNA functionalized QCdSe integrated miniaturized system hasmore » been used to monitor target complementary DNA concentration by measuring the interfacial charge transfer resistance via hybridization. The presence of complementary DNA in buffer solution significantly results in decreased electro-conductivity of the interface due to presence of a charge barrier for transport of the redox probe ions. The microfluidic DNA biosensor exhibits improved linearity in the concentration range of 10{sup −15} M to 10{sup −11} M.« less

Low energy electron catalyst: the electronic origin of catalytic strategies.

PubMed

Davis, Daly; Sajeev, Y

2016-10-12

Using a low energy electron (LEE) as a catalyst, the electronic origin of the catalytic strategies corresponding to substrate selectivity, reaction specificity and reaction rate enhancement is investigated for a reversible unimolecular elementary reaction. An electronic energy complementarity between the catalyst and the substrate molecule is the origin of substrate selectivity and reaction specificity. The electronic energy complementarity is induced by tuning the electronic energy of the catalyst. The energy complementarity maximizes the binding forces between the catalyst and the molecule. Consequently, a new electronically metastable high-energy reactant state and a corresponding new low barrier reaction path are resonantly created for a specific reaction of the substrate through the formation of a catalyst-substrate transient adduct. The LEE catalysis also reveals a fundamental structure-energy correspondence in the formation of the catalyst-substrate transient adduct. Since the energy complementarities corresponding to the substrate molecules of the forward and the backward steps of the reversible reactions are not the same due to their structural differences, the LEE catalyst exhibits a unique one-way catalytic strategy, i.e., the LEE catalyst favors the reversible reaction more effectively in one direction. A characteristic stronger binding of the catalyst to the transition state of the reaction than in the initial reactant state and the final product state is the molecular origin of barrier lowering.

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

PubMed

Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri

2018-02-01

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The effects of substrate pre-treatment on anaerobic digestion systems: a review.

PubMed

Carlsson, My; Lagerkvist, Anders; Morgan-Sagastume, Fernando

2012-09-01

Focus is placed on substrate pre-treatment in anaerobic digestion (AD) as a means of increasing biogas yields using today's diversified substrate sources. Current pre-treatment methods to improve AD are being examined with regard to their effects on different substrate types, highlighting approaches and associated challenges in evaluating substrate pre-treatment in AD systems and its influence on the overall system of evaluation. WWTP residues represent the substrate type that is most frequently assessed in pre-treatment studies, followed by energy crops/harvesting residues, organic fraction of municipal solid waste, organic waste from food industry and manure. The pre-treatment effects are complex and generally linked to substrate characteristics and pre-treatment mechanisms. Overall, substrates containing lignin or bacterial cells appear to be the most amendable to pre-treatment for enhancing AD. Approaches used to evaluate AD enhancement in different systems is further reviewed and challenges and opportunities for improved evaluations are identified. Copyright © 2012 Elsevier Ltd. All rights reserved.

Process for metallization of a substrate by irradiative curing of a catalyst applied thereto

DOEpatents

Chen, Ken S.; Morgan, William P.; Zich, John L.

1999-01-01

An improved additive process for metallization of substrates is described whereby a catalyst solution is applied to a surface of a substrate. Metallic catalytic clusters can be formed in the catalyst solution on the substrate surface by irradiating the substrate. Electroless plating can then deposit metal onto the portion of the substrate surface having metallic clusters. Additional metallization thickness can be obtained by electrolytically plating the substrate surface after the electroless plating step.

Process for metallization of a substrate by curing a catalyst applied thereto

DOEpatents

Chen, Ken S.; Morgan, William P.; Zich, John L.

2002-10-08

An improved additive process for metallization of substrates is described whereby a catalyst solution is applied to a surface of a substrate. Metallic catalytic clusters can be formed in the catalyst solution on the substrate surface by heating the substrate. Electroless plating can then deposit metal onto the portion of the substrate surface coated with catalyst solution. Additional metallization thickness can be obtained by electrolytically plating the substrate surface after the electroless plating step.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

PubMed

Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

2011-08-10

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

PubMed Central

2011-01-01

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

PubMed Central

Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

2016-01-01

Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Biochemistry students' ideas about how an enzyme interacts with a substrate.

PubMed

Linenberger, Kimberly J; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

PubMed

Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min

2016-03-01

In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.

Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development.

PubMed

Merlaen, Britt; De Keyser, Ellen; Van Labeke, Marie-Christine

2018-01-01

The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant-water relations in the commercial strawberry ( Fragaria x ananassa ). Aquaporins are water channel proteins that allow water to cross (intra)cellular membranes. In Fragaria x ananassa , few of them have been identified hitherto, hampering the exploration of the water transport regulation at cellular level. Here, we present new aquaporin coding sequences belonging to different subclasses: plasma membrane intrinsic proteins subtype 1 and subtype 2 (PIP1 and PIP2) and tonoplast intrinsic proteins (TIP). The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues. Substrate-specific signature sequences (SSSSs) and specificity-determining positions (SDPs) predict the substrate specificity of each new aquaporin. Expression profiling in leaves, petioles and developing fruits reveals distinct patterns, even within the same (sub)class. Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression. Both upregulation and downregulation during fruit ripening occur. Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same (sub)class.

h-BN Nanosheets as 2D Substrates to Load 0D Fe3O4 Nanoparticles: A Hybrid Anode Material for Lithium-Ion Batteries.

PubMed

Duan, Zhi-Qiang; Liu, Yi-Tao; Xie, Xu-Ming; Ye, Xiong-Ying; Zhu, Xiao-Dong

2016-03-18

h-BN, as an isoelectronic analogue of graphene, has improved thermal mechanical properties. Moreover, the liquid-phase production of h-BN is greener since harmful oxidants/reductants are unnecessary. Here we report a novel hybrid architecture by employing h-BN nanosheets as 2D substrates to load 0D Fe3O4 nanoparticles, followed by phenol/formol carbonization to form a carbon coating. The resulting carbon-encapsulated h-BN@Fe3O4 hybrid architecture exhibits synergistic interactions: 1) The h-BN nanosheets act as flexible 2D substrates to accommodate the volume change of the Fe3O4 nanoparticles; 2) The Fe3O4 nanoparticles serve as active materials to contribute to a high specific capacity; and 3) The carbon coating not only protects the hybrid architecture from deformation but also keeps the whole electrode highly conductive. The synergistic interactions translate into significantly enhanced electrochemical performances, laying a basis for the development of superior hybrid anode materials. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

PubMed

Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

2012-05-01

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

Direct measurement of catalase activity in living cells and tissue biopsies

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

Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Usingmore » catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear demonstration of the applicability in cancer cells and aging animal tissues.« less

An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase, a key enzyme in artemisinin biosynthesis.

PubMed

Eslami, Habib; Mohtashami, Seyed Kaveh; Basmanj, Maryam Taghavi; Rahati, Maryam; Rahimi, Hamzeh

2017-07-01

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg 2+ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519-532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg 2+ . Such detailed understanding will probably help to design an improved enzyme.

High acetone-butanol-ethanol production in pH-stat co-feeding of acetate and glucose.

PubMed

Gao, Ming; Tashiro, Yukihiro; Wang, Qunhui; Sakai, Kenji; Sonomoto, Kenji

2016-08-01

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Microbial anodic consortia fed with fermentable substrates in microbial electrolysis cells: Significance of microbial structures.

PubMed

Flayac, Clément; Trably, Eric; Bernet, Nicolas

2018-05-28

Microbial community structure of anodic biofilms plays a key role in bioelectrochemical systems (BESs). When ecosystems are used as inocula, many bacterial species having interconnected ecological interactions are present. The aim of the present study was to identify these interactions for the conversion of single substrates into electrical current. Dual-chamber reactors were inoculated with activated sludge and fed in batch mode with acetate, lactate, butyrate and propionate at 80 mMe - equivalents in quadruplicate. Analyses of biofilms and planktonic microbial communities showed that the anodic biofilms were mainly dominated by the Geobacter genus (62.4% of the total sequences). At the species level, Geobacter sulfurreducens was dominant in presence of lactate and acetate, while Geobacter toluenoxydans and Geobacter pelophilus were dominant with butyrate and propionate as substrates. These results indicate for the first time a specificity within the Geobacter genus towards the electron donor, suggesting a competitive process for electrode colonization and the implementations of syntrophic interactions for complete oxidation of substrates such as propionate and butyrate. All together, these results provide a new insight into the ecological relationships within electroactive biofilms and suggest eco-engineering perspectives to improve the performances of BESs. Copyright © 2018 Elsevier B.V. All rights reserved.

Field emission from amorphous carbon films grown by electrochemical deposition using methanol liquid

NASA Astrophysics Data System (ADS)

Kiyota, H.; Higashi, M.; Kurosu, T.; Iida, M.

2006-05-01

The field emission from an amorphous carbon (a-C) film grown by electrochemical deposition has been studied. The deposition of the a-C film was accomplished by applying a direct-current potential to a substrate that was immersed in methanol. Both scanning electron microscopy and Raman results indicate that smooth and homogeneous a-C films are grown on specific substrates such as Ti and Al. Field emission measurements demonstrate excellent emission properties such as threshold fields as low as 5 V/μm. Enhancement factors are estimated to be in the range of 1300-1500; these are attributed to local field enhancements around sp2 carbon clusters that are embedded in the a-C films. Emission properties of a-C films grown on Si exhibit a current saturation under higher applied fields. These saturation characteristics are explained by effects of a potential barrier at the interface between the a-C film and the substrate. The interface barrier is reduced by formation of the Ti interfacial layer, suggesting that the formation of TiC decreases the contact resistance between the substrate and the a-C film. Therefore, an approach to use carbide formation at the interface is verified as useful to improve the emission properties of a-C films.

Application of N-doped graphene modified carbon ionic liquid electrode for direct electrochemistry of hemoglobin.

PubMed

Sun, Wei; Dong, Lifeng; Deng, Ying; Yu, Jianhua; Wang, Wencheng; Zhu, Qianqian

2014-06-01

Nitrogen-doped graphene (NG) was synthesized and used for the investigation on direct electrochemistry of hemoglobin (Hb) with a carbon ionic liquid electrode as the substrate electrode. Due to specific characteristics of NG such as excellent electrocatalytic property and large surface area, direct electron transfer of Hb was realized with enhanced electrochemical responses appearing. Electrochemical behaviors of Hb on the NG modified electrode were carefully investigated with the electrochemical parameters calculated. The Hb modified electrode exhibited excellent electrocatalytic reduction activity toward different substrates, such as trichloroacetic acid and H2O2, with wider dynamic range and lower detection limit. These findings show that NG can be used for the preparation of chemically modified electrodes with improved performance and has potential applications in electrochemical sensing. Copyright © 2014 Elsevier B.V. All rights reserved.

SVGA and XGA active matrix microdisplays for head-mounted applications

NASA Astrophysics Data System (ADS)

Alvelda, Phillip; Bolotski, Michael; Brown, Imani L.

2000-03-01

The MicroDisplay Corporation's liquid crystal on silicon (LCOS) display devices are based on the union of several technologies with the extreme integration capability of conventionally fabricated CMOS substrates. The fast liquid crystal operation modes and new scalable high-performance pixel addressing architectures presented in this paper enable substantially improved color, contrast, and brightness while still satisfying the optical, packaging, and power requirements of portable applications. The entire suite of MicroDisplay's technologies was devised to create a line of mixed-signal application-specific integrated circuits (ASICs) in single-chip display systems. Mixed-signal circuits can integrate computing, memory, and communication circuitry on the same substrate as the display drivers and pixel array for a multifunctional complete system-on-a-chip. System-on-a-chip benefits also include reduced head supported weight requirements through the elimination of off-chip drive electronics.

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

PubMed

Tyagi, Sadhna; Pleiss, Juergen

2006-06-25

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

PubMed Central

Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

Green roofs for a drier world: effects of hydrogel amendment on substrate and plant water status.

PubMed

Savi, Tadeja; Marin, Maria; Boldrin, David; Incerti, Guido; Andri, Sergio; Nardini, Andrea

2014-08-15

Climate features of the Mediterranean area make plant survival over green roofs challenging, thus calling for research work to improve water holding capacities of green roof systems. We assessed the effects of polymer hydrogel amendment on the water holding capacity of a green roof substrate, as well as on water status and growth of Salvia officinalis. Plants were grown in green roof experimental modules containing 8 cm or 12 cm deep substrate (control) or substrate mixed with hydrogel at two different concentrations: 0.3 or 0.6%. Hydrogel significantly increased the substrate's water content at saturation, as well as water available to vegetation. Plants grown in 8 cm deep substrate mixed with 0.6% of hydrogel showed the best performance in terms of water status and membrane integrity under drought stress, associated to the lowest above-ground biomass. Our results provide experimental evidence that polymer hydrogel amendments enhance water supply to vegetation at the establishment phase of a green roof. In particular, the water status of plants is most effectively improved when reduced substrate depths are used to limit the biomass accumulation during early growth stages. A significant loss of water holding capacity of substrate-hydrogel blends was observed after 5 months from establishment of the experimental modules. We suggest that cross-optimization of physical-chemical characteristics of hydrogels and green roof substrates is needed to improve long term effectiveness of polymer-hydrogel blends. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of substrate quality on IC performance and yields

NASA Technical Reports Server (NTRS)

Thomas, R. N.

1981-01-01

The development of silicon and gallium arsenide crystal growth for the production of large diameter substrates are discussed. Large area substrates of significantly improved compositional purity, dopant distribution and structural perfection on a microscopic as well as macroscopic scale are important requirements. The exploratory use of magnetic fields to suppress convection effects in Czochralski crystal growth is addressed. The growth of large crystals in space appears impractical at present however the efforts to improve substrate quality could benefit from the experiences gained in smaller scale growth experiments conducted in the zero gravity environment of space.

Powder-in-tube and thick-film methods of fabricating high temperature superconductors having enhanced biaxial texture

DOEpatents

Goyal, Amit; Kroeger, Donald M.

2003-11-11

A method for forming an electronically active biaxially textured article includes the steps of providing a substrate having a single crystal metal or metal alloy surface, deforming the substrate to form an elongated substrate surface having biaxial texture and depositing an epitaxial electronically active layer on the biaxially textured surface. The method can include at least one annealing step after the deforming step to produce the biaxially textured substrate surface. The invention can be used to form improved biaxially textured articles, such as superconducting wire and tape articles having improved J.sub.c values.

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Microstructure and Antiwear Property of Laser Cladding Ni–Co Duplex Coating on Copper

PubMed Central

Wang, Yiyong; Liang, Zhipeng; Zhang, Junwei; Ning, Zhe; Jin, Hui

2016-01-01

Ni–Co duplex coatings were cladded onto Cu to improve the antiwear properties of Cu products. Prior to laser cladding, n-Al2O3/Ni layers were introduced as interlayers between laser cladding coatings and Cu substrates to improve the laser absorptivity of these substrates and ensure defect-free laser cladding coatings. The structure and morphology of the coatings were characterized by scanning electron microscopy and optical microscopy, and the phases of the coatings were analyzed by X-ray diffraction. Their hardness was measured using a microhardness tester. Experimental results showed that defect-free composite coatings were obtained and that the coatings were metallurgically bonded to the substrates. The surface of the Ni–Co duplex coatings comprised a Co-based solid solution, Cr7C3, (Fe,Ni)23C6, and other strengthening phases. The microhardness and wear resistance of the duplex coatings were significantly improved compared with the Cu substrates. The average microhardness of the cladded coatings was 845.6 HV, which was approximately 8.2 times greater than that of the Cu substrates (102.6 HV). The volume loss of the Cu substrates was approximately 7.5 times greater than that of the Ni–Co duplex coatings after 60 min of sliding wear testing. The high hardness of and lack of defects in the Ni–Co duplex coatings reduced the plastic deformation and adhesive wear of the Cu substrates, resulting in improved wear properties. PMID:28773755

High yield growth of patterned vertically aligned carbon nanotubes using inkjet-printed catalyst.

PubMed

Beard, James D; Stringer, Jonathan; Ghita, Oana R; Smith, Patrick J

2013-10-09

This study reports on the fabrication of vertically aligned carbon nanotubes localized at specific sites on a growth substrate by deposition of a nanoparticle suspension using inkjet printing. Carbon nanotubes were grown with high yield as vertically aligned forests to a length of approximately 400 μm. The use of inkjet printing for catalyst fabrication considerably improves the production rate of vertically aligned patterned nanotube forests compared with conventional patterning techniques, for example, electron beam lithography or photolithography.

Citrobacter diversus ULA-27 beta-lactamases. Improved purification and general properties.

PubMed Central

Amicosante, G; Oratore, A; Franceschini, N; Maccarrone, M; Strom, R; Galleni, M; Frère, J M

1988-01-01

Two chromosome-encoded beta-lactamases have been purified from Citrobacter diversus ULA-27. They exhibited slightly different isoelectric points (6.8 and 6.2) and very similar Mr values (congruent to 29,000). Their specificity spectrum was rather wide, since they hydrolysed some cephalosporins with kcat: values similar to those observed with the best penicillin substrates. Cloxacillin, methicillin and imipenem were hydrolysed very slowly. Hydrolysis of azthreonam could not be detected. Images Fig. 3. Fig. 4. PMID:3264152

Recent advances in rational approaches for enzyme engineering

PubMed Central

Steiner, Kerstin; Schwab, Helmut

2012-01-01

Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications. PMID:24688651

Effects of substrate concentrations on the growth of heterotrophic bacteria and algae in secondary facultative ponds.

PubMed

Kayombo, S; Mbwette, T S A; Katima, J H Y; Jorgensen, S E

2003-07-01

This paper presents the effect of substrate concentration on the growth of a mixed culture of algae and heterotrophic bacteria in secondary facultative ponds (SFPs) utilizing settled domestic sewage as a sole source of organic carbon. The growth of the mixed culture was studied at the concentrations ranging between 200 and 800 mg COD/l in a series of batch chemostat reactors. From the laboratory data, the specific growth rate (micro) was determined using the modified Gompertz model. The maximum specific growth rate ( micro(max)) and half saturation coefficients (K(s)) were calculated using the Monod kinetic equation. The maximum observed growth rate ( micro(max)) for heterotrophic bacteria was 3.8 day(-1) with K(s) of 200 mg COD/l. The micro(max) for algal biomass based on suspended volatile solids was 2.7 day(-1) with K(s) of 110 mg COD/l. The micro(max) of algae based on the chlorophyll-a was 3.5 day(-1) at K(s) of 50mg COD/l. The observed specific substrate removal by heterotrophic bacteria varied between the concentrations of substrate used and the average value was 0.82 (mg COD/mg biomass). The specific substrate utilization rate in the bioreactors was direct proportional to the specific growth rate. Hence, the determined Monod kinetic parameters are useful for the definition of the operation of SFPs.

A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity.

PubMed

Buryska, Tomas; Babkova, Petra; Vavra, Ondrej; Damborsky, Jiri; Prokop, Zbynek

2018-01-15

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications. IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols. Copyright © 2018 American Society for Microbiology.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Exploration of CIGAS Alloy System for Thin-Film Photovoltaics on Novel Lightweight and Flexible Substrates

NASA Technical Reports Server (NTRS)

Woods, Lawrence M.; Kalla, Ajay; Ribelin, Rosine

2007-01-01

Thin-film photovoltaics (TFPV) on lightweight and flexible substrates offer the potential for very high solar array specific power (W/kg). ITN Energy Systems, Inc. (ITN) is developing flexible TFPV blanket technology that has potential for specific power greater than 2000 W/kg (including space coatings) that could result in solar array specific power between 150 and 500 W/kg, depending on array size, when mated with mechanical support structures specifically designed to take advantage of the lightweight and flexible substrates.(1) This level of specific power would far exceed the current state of the art for spacecraft PV power generation, and meet the needs for future spacecraft missions.(2) Furthermore the high specific power would also enable unmanned aircraft applications and balloon or high-altitude airship (HAA) applications, in addition to modular and quick deploying tents for surface assets or lunar base power, as a result of the high power density (W/sq m) and ability to be integrated into the balloon, HAA or tent fabric. ITN plans to achieve the high specific power by developing single-junction and two-terminal monolithic tandem-junction PV cells using thin-films of high-efficiency and radiation resistant CuInSe2 (CIS) partnered with bandgap-tunable CIS-alloys with Ga (CIGS) or Al (CIAS) on novel lightweight and flexible substrates. Of the various thin-film technologies, single-junction and radiation resistant CIS and associated alloys with gallium, aluminum and sulfur have achieved the highest levels of TFPV device performance, with the best efficiency reaching 19.5% under AM1.5 illumination conditions and on thick glass substrates.(3) Thus, it is anticipated that single- and tandem-junction devices with flexible substrates and based on CIS and related alloys will achieve the highest levels of thin-film space and HAA solar array performance.

Colloidal spray method for low cost thin coating deposition

DOEpatents

Pham, Ai-Quoc; Glass, Robert S.; Lee, Tae H.

2005-01-25

A dense or porous coating of material is deposited onto a substrate by forcing a colloidal suspension through an ultrasonic nebulizer and spraying a fine mist of particles in a carrier medium onto a sufficiently heated substrate. The spraying rate is essentially matched to the evaporation rate of the carrier liquid from the substrate to produce a coating that is uniformly distributed over the surface of the substrate. Following deposition to a sufficient coating thickness, a single sintering step may be used to produce a dense ceramic coating. Using this method, coatings ranging in thickness from about one to several hundred microns can be obtained. By using a plurality of compounds in the colloidal suspension, coatings of mixed composition can be obtained. By using a plurality of solutions and separate pumps and a single or multiple ultrasonic nebulizer(s), and varying the individual pumping rates and/or the concentrations of the solutions, a coating of mixed and discontinuously graded (e.g., stepped) or continuously graded layers may be obtained. This method is particularly useful for depositing ceramic coatings. Dense ceramic coating materials on porous substrates are useful in providing improved electrode performance in devices such as high power density solid oxide fuel cells. Dense ceramic coatings obtained by the invention are also useful for gas turbine blade coatings, sensors, steam electrolyzers, etc. The invention has general use in preparation of systems requiring durable and chemically resistant coatings, or coatings having other specific chemical or physical properties.

Colloidal spray method for low cost thin coating deposition

DOEpatents

Pham, Ai-Quoc; Glass, Robert S.; Lee, Tae H.

2002-01-01

A dense or porous coating of material is deposited onto a substrate by forcing a colloidal suspension through an ultrasonic nebulizer and spraying a fine mist of particles in a carrier medium onto a sufficiently heated substrate. The spraying rate is essentially matched to the evaporation rate of the carrier liquid from the substrate to produce a coating that is uniformly distributed over the surface of the substrate. Following deposition to a sufficient coating thickness, a single sintering step may be used to produce a dense ceramic coating. Using this method, coatings ranging in thickness from about one to several hundred microns can be obtained. By using a plurality of compounds in the colloidal suspension, coatings of mixed composition can be obtained. By using a plurality of solutions and separate pumps and a single or multiple ultrasonic nebulizer(s), and varying the individual pumping rates and/or the concentrations of the solutions, a coating of mixed and discontinuously graded (e.g., stepped) or continuously graded layers may be obtained. This method is particularly useful for depositing ceramic coatings. Dense ceramic coating materials on porous substrates are useful in providing improved electrode performance in devices such as high power density solid oxide fuel cells. Dense ceramic coatings obtained by the invention are also useful for gas turbine blade coatings, sensors, steam electrolyzers, etc. The invention has general use in preparation of systems requiring durable and chemically resistant coatings, or coatings having other specific chemical or physical properties.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Kinetic modeling of lactic acid production from batch submerged fermentation of cheese whey

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

Tango, M.S.A.; Ghaly, A.E.

1999-12-01

A kinetic model for the production of lactic acid through batch submerged fermentation of cheese whey using Lactobacillus helveticus was developed. The model accounts for the effect of substrate limitation, substrate inhibition, lactic acid inhibition, maintenance energy and cell death on the cell growth, substrate utilization, and lactic acid production during the fermentation process. The model was evaluated using experimental data from Tango and Ghaly (1999). The predicted results obtained from the model compared well with experimental (R{sup 2} = 0.92--0.98). The model was also used to investigate the effect of the initial substrate concentration on the lag period, fermentationmore » time, specific growth rate, and cell productivity during batch fermentation. The maximum specific growth rate ({micro}{sub m}), the saturation constant (K{sub S}), the substrate inhibition constant (K{sub IS}), and the lactic acid inhibition constant (K{sub IP}) were found to be 0.25h{sup {minus}1}, 0.9 g/L, 250.0 g/L, and 60.0 g/L, respectively. High initial lactose concentration in cheese whey reduced both the specific growth rate and substrate utilization rate due to the substrate inhibition phenomenon. The maximum lactic acid production occurred at about 100 g/L initial lactose concentration after 40 h of fermentation. The maximum lactic acid concentration above which Lactobacillus helveticus did not grow was found to be 80.0 g/L.« less

Toward Improved Lifetimes of Organic Solar Cells under Thermal Stress: Substrate-Dependent Morphological Stability of PCDTBT:PCBM Films and Devices.

PubMed

Li, Zhe; Ho Chiu, Kar; Shahid Ashraf, Raja; Fearn, Sarah; Dattani, Rajeev; Cheng Wong, Him; Tan, Ching-Hong; Wu, Jiaying; Cabral, João T; Durrant, James R

2015-10-15

Morphological stability is a key requirement for outdoor operation of organic solar cells. We demonstrate that morphological stability and lifetime of polymer/fullerene based solar cells under thermal stress depend strongly on the substrate interface on which the active layer is deposited. In particular, we find that the stability of benchmark PCDTBT/PCBM solar cells under modest thermal stress is substantially increased in inverted solar cells employing a ZnO substrate compared to conventional devices employing a PSS substrate. This improved stability is observed to correlate with PCBM nucleation at the 50 nm scale, which is shown to be strongly influenced by different substrate interfaces. Employing this approach, we demonstrate remarkable thermal stability for inverted PCDTBT:PC70BM devices on ZnO substrates, with negligible (<2%) loss of power conversion efficiency over 160 h under 85 °C thermal stress and minimal thermally induced "burn-in" effect. We thus conclude that inverted organic solar cells, in addition to showing improved environmental stability against ambient humidity exposure as widely reported previously, can also demonstrate enhanced morphological stability. As such we show that the choice of suitable substrate interfaces may be a key factor in achieving prolonged lifetimes for organic solar cells under thermal stress conditions.

Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

PubMed Central

Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.

2001-01-01

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors. PMID:11533208

Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

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

Kumar, Manoj

2011-05-09

These are a set of slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

PubMed

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-05-01

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from Mycobacterium tuberculosis

PubMed Central

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. PMID:23843969

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

PubMed

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

[Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus Bathypolypus arcticus].

PubMed

Basova, I N; Iagodina, O V

2012-01-01

Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.

Study on effect of plasma surface treatments for diamond deposition by DC arc plasmatron.

PubMed

Kang, In-Je; Joa, Sang-Beom; Lee, Heon-Ju

2013-11-01

To improve the thermal conductivity and wear resistance of ceramic materials in the field of renewable energy technologies, diamond coating by plasma processing has been carried out in recent years. This study's goal is to improve diamond deposition on Al2O3 ceramic substrates by plasma surface treatments. Before diamond deposition was carried out in a vacuum, plasma surface treatments using Ar gas were conducted to improve conditions for deposition. We also conducted plasma processing for diamond deposition on Al2O3 ceramic substrates using a DC arc Plasmatron. The Al2O3 ceramic substrates with diamond film (5 x 15 mm2), were investigated by SEM (Scanning Electron Microscopy), AFM (Atomic Force Microscopy) and XRD (X-ray Diffractometer). Then, the C-H stretching of synthetic diamond films by FTIR (Fourier Transform Infrared Spectroscopy) was studied. We identified nanocrystalline diamond films on the Al2O3 ceramic substrates. The results showed us that the deposition rate of diamond films was 2.3 microm/h after plasma surface treatments. Comparing the above result with untreated ceramic substrates, the deposition rate improved with the surface roughness of the deposited diamond films.

Structure and Activity Analyses of Escherichia coli K-12 NagD Provide Insight into the Evolution of Biochemical Function in the Haloakanoic Acid Dehlogenase Superfamily

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

Tremblay,L.; Dunaway-Mariano, D.; Allen, K.

2006-01-01

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia colimore » K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 Angstroms with R{sub work} = 19.8% and R{sub free} = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with kcat/Km = 3.12 x 10{sup 4} and 1.28 x 10{sup 4} {micro}M{sup -1} s{sup -1} for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k{sub cat}/K{sub m}) are low (1 x 10{sup 4} M{sup -1} s{sup -1}) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.« less

Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.

PubMed

Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung

2015-06-01

N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.

Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics.

PubMed

Cullis, Ian F; Saddler, John N; Mansfield, Shawn D

2004-02-20

Previous optimization strategies for the bioconversion of lignocellulosics by steam explosion technologies have focused on the effects of temperature, pH, and treatment time, but have not accounted for changes in severity brought about by properties inherent in the starting feedstock. Consequently, this study evaluated the effects of chip properties, feedstock size (40-mesh, 1.5 x 1.5 cm, 5 x 5 cm), and moisture content (12% and 30%) on the overall bioconversion process, and more specifically on the efficacy of removal of recalcitrant lignin from the lignocellulosic substrates following steam explosion. Increasing chip size resulted in an improvement in the solids recovery, with concurrent increases in the water soluble, hemicellulose-derived sugar recovery (7.5%). This increased recovery is a result of a decrease in the "relative severity" of the pretreatment as chip size increases. Additionally, the decreased relative severity minimized the condensation of the recalcitrant residual lignin and therefore increased the efficacy of peroxide fractionation, where a 60% improvement in lignin removal was possible with chips of larger initial size. Similarly, increased initial moisture content reduced the relative severity of the pretreatment, generating improved solids and hemicellulose-derived carbohydrate recovery. Both increased chip size and higher initial moisture content results in a substrate that performs better during peroxide delignification, and consequently enzymatic hydrolysis. Furthermore, a post steam-explosion refining step increased hemicellulose-derived sugar recovery and was most effectively delignified (to as low as 6.5%). The refined substrate could be enzymatically hydrolyzed to very high levels (98%) and relatively fast rates (1.23 g/L/h). Copyright 2004 Wiley Periodicals, Inc.

A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

PubMed Central

Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.

2012-01-01

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

PubMed

Dzyadevych, Sergei V; Soldatkin, Alexey P; Korpan, Yaroslav I; Arkhypova, Valentyna N; El'skaya, Anna V; Chovelon, Jean-Marc; Martelet, Claude; Jaffrezic-Renault, Nicole

2003-10-01

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.

Biomimicry in biomedical research

PubMed Central

Zhang, Ge

2012-01-01

Biomimicry (literally defined as the imitation of life or nature) has sparked a variety of human innovations and inspired countless cutting-edge designs. From spider silk-made artificial skin to lotus leaf-inspired self-cleaning materials, biomimicry endeavors to solve human problems. Biomimetic approaches have contributed significantly to advances biomedical research during recent years. Using polyacrylamide gels to mimic the elastic modulus of different biological tissues, Disher’s lab has directed meschymal stem cell differentiation into specific lineages.1 They have shown that soft substrates mimicking the elastic modulus of brain tissues (0.1~1 kPa) were neurogenic, substrates of intermediate elastic modulus mimicking muscle (8 ~17 kPa) were myogenic, and substrates with bone-like elastic modulus (25~40 kPa) were osteogenic. This work represents a novel way to regulate the fate of stem cells and exerts profound influence on stem cell research. Biomimcry also drives improvements in tissue engineering. Novel scaffolds have been designed to capture extracellular matrix-like structures, binding of ligands, sustained release of cytokines, and mechanical properties intrinsic to specific tissues for tissue engineering applications.2,3 For example, tissue engineering skin grafts have been designed to mimic the cell composition and layered structure of native skin.4 Similarly, in the field of regenerative medicine, researchers aim to create biomimetic scaffolds to mimic the properties of a native stem cell environment (niche) to dynamically interact with the entrapped stem cells and direct their response.5 PMID:23275257

Substrate Specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa

PubMed Central

Masuda, Nobuhisa; Sakagawa, Eiko; Ohya, Satoshi; Gotoh, Naomasa; Tsujimoto, Hideto; Nishino, Takeshi

2000-01-01

To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the β-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously. PMID:11083635

[Substrate specifity in Amoeba proteus].

PubMed

Sopina, V A

2006-01-01

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.

On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways

NASA Technical Reports Server (NTRS)

Lazcano, A.; Diaz-Villagomez, E.; Mills, T.; Oro, J.

1995-01-01

The most frequently invoked explanation for the origin of metabolic pathways is the retrograde evolution hypothesis. In contrast, according to the so-called 'patchwork' theory, metabolism evolved by the recruitment of relatively inefficient small enzymes of broad specificity that could react with a wide range of chemically related substrates. In this paper it is argued that both sequence comparisons and experimental results on enzyme substrate specificity support the patchwork assembly theory. The available evidence supports previous suggestions that gene duplication events followed by a gradual neoDarwinian accumulation of mutations and other minute genetic changes lead to the narrowing and modification of enzyme function in at least some primordial metabolic pathways.

MnO2-Based Electrochemical Supercapacitors on Flexible Carbon Substrates

NASA Astrophysics Data System (ADS)

Tadjer, Marko J.; Mastro, Michael A.; Rojo, José M.; Mojena, Alberto Boscá; Calle, Fernando; Kub, Francis J.; Eddy, Charles R.

2014-04-01

Manganese dioxide films were grown on large area flexible carbon aerogel substrates. Characterization by x-ray diffraction confirmed α-MnO2 growth. Three types of films were compared as a function of hexamethylenetetramine (HMTA) concentration during growth. The highest concentration of HM TA produced MnO2 flower-like films, as observed by scanning electron microscopy, whose thickness and surface coverage lead to both a higher specific capacitance and higher series resistance. Specific capacitance was measured to be 64 F/g using a galvanostatic setup, compared to the 47 F/g-specific capacitance of the carbon aerogel substrate. Such supercapacitor devices can be fabricated on large area sheets of carbon aerogel to achieve high total capacitance.

Masks For Deposition Of Aspherical Optical Surfaces

NASA Technical Reports Server (NTRS)

Rogers, John R.; Martin, John D.

1992-01-01

Masks of improved design developed for use in fabrication of aspherical, rotationally symmetrical surfaces of mirrors, lenses, and lens molds by evaporative deposition onto rotating substrates. In deposition chamber, source and mask aligned with axis of rotation of substrate. Mask shadows source of rotating substrate. Azimuthal opening (as function of radius) in mask proportional to desired thickness (as function of radius) to which material deposited on substrate. Combination of improved masks and modern coating chambers provides optical surfaces comparable or superior to those produced by conventional polishing, computer-controlled polishing, replication from polished molds, and diamond turning, at less cost in material, labor, and capital expense.

Graphene electrodes for stimulation of neuronal cells

NASA Astrophysics Data System (ADS)

Koerbitzer, Berit; Krauss, Peter; Nick, Christoph; Yadav, Sandeep; Schneider, Joerg J.; Thielemann, Christiane

2016-06-01

Graphene has the ability to improve the electrical interface between neuronal cells and electrodes used for recording and stimulation purposes. It provides a biocompatible coating for common electrode materials such as gold and improves the electrode properties. Graphene electrodes are also prepared on SiO2 substrate to benefit from its optical properties like transparency. We perform electrochemical and Raman characterization of gold electrodes with graphene coating and compare them with graphene on SiO2 substrate. It was found that the substrate plays an important role in the performance of graphene and show that graphene on SiO2 substrate is a very promising material combination for stimulation electrodes.

Oyster Culinary-Medicinal Mushroom, Pleurotus ostreatus (Higher Basidiomycetes), Growth in Grain-Based Diet Improves Broiler Chicken Production.

PubMed

Santos, Mateus P; Marcante, Rafael C; Santana, Thiago T; Tanaka, Henrique S; Funari, Pascoal; Alberton, Luiz R; Faria, Eliete V; Valle, Juliana S; Colauto, Nelson B; Linde, Giani A

2015-01-01

Many alternative compounds have been tested to improve poultry performance but few of them have previously used mycelial-colonized substrate to partially replace standard diet in broiler chickens. The objective of this study was to evaluate broiler chicken production, health, and meat sensory characteristics, with partial replacement of the standard diet by Pleurotus ostreatus-colonized substrate. One hundred fifty 1-day-old male Cobb chicks were given standard diet partially replaced by 0, 5, 10, 100, or 200 g·kg⁻¹ of P. ostreatus-colonized substrate and randomly distributed into five treatments. Each treatment had three replicates, with 10 birds per replicate, totaling 30 birds. The replacement of the standard diet by 10 g·kg⁻¹ of colonized substrate increased (P≤0.05) chicken body mass up to 57% at 21 days, and up to 28% at 42 days. In general, partial replacement of standard diet by colonized substrate increased hematocrits and typical lymphocytes, and reduced low density lipoproteins. Also, it reduced chicken production period up to 21% and there is no meat taste alteration. The use of P. ostreatus-colonized substrate in chicken feeding is an alternative method to improve broiler chicken production.

Nickel enhanced graphene growth directly on dielectric substrates by molecular beam epitaxy

NASA Astrophysics Data System (ADS)

Wofford, Joseph M.; Speck, Florian; Seyller, Thomas; Lopes, Joao Marcelo J.; Riechert, Henning

2016-07-01

The efficacy of Ni as a surfactant to improve the crystalline quality of graphene grown directly on dielectric Al2O3(0001) substrates by molecular beam epitaxy is examined. Simultaneously exposing the substrate to a Ni flux throughout C deposition at 950 °C led to improved charge carrier mobility and a Raman spectrum indicating less structural disorder in the resulting nanocrystalline graphene film. X-ray photoelectron spectroscopy confirmed that no residual Ni could be detected in the film and showed a decrease in the intensity of the defect-related component of the C1s level. Similar improvements were not observed when a lower substrate temperature (850 °C) was used. A close examination of the Raman spectra suggests that Ni reduces the concentration of lattice vacancies in the film, possibly by catalytically assisting adatom incorporation.

Computational methods in metabolic engineering for strain design.

PubMed

Long, Matthew R; Ong, Wai Kit; Reed, Jennifer L

2015-08-01

Metabolic engineering uses genetic approaches to control microbial metabolism to produce desired compounds. Computational tools can identify new biological routes to chemicals and the changes needed in host metabolism to improve chemical production. Recent computational efforts have focused on exploring what compounds can be made biologically using native, heterologous, and/or enzymes with broad specificity. Additionally, computational methods have been developed to suggest different types of genetic modifications (e.g. gene deletion/addition or up/down regulation), as well as suggest strategies meeting different criteria (e.g. high yield, high productivity, or substrate co-utilization). Strategies to improve the runtime performances have also been developed, which allow for more complex metabolic engineering strategies to be identified. Future incorporation of kinetic considerations will further improve strain design algorithms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plasma-assisted nitrogen doping of VACNTs for efficiently enhancing the supercapacitor performance

NASA Astrophysics Data System (ADS)

Mashayekhi, Alireza; Hosseini, Seyed Mahmoud; Hassanpour Amiri, Morteza; Namdar, Naser; Sanaee, Zeinab

2016-06-01

Nitrogen doping of vertically aligned carbon nanotubes (VACNTs) using plasma-enhanced chemical vapour deposition has been investigated to improve the supercapacitance performance of CNTs. Incorporating electrochemical measurements on the open-ended nitrogen-doped CNTs, showed the achievement of 6 times improvement in the capacitance value. For nitrogen-doped CNTs on silicon substrate, specific capacitance of 60 F g-1 was obtained in 0.5 M KCl solution, with capacity retention ratio above 90 % after cycled at 0.1 A g-1 for 5000 cycles. Using this sample, a symmetric supercapacitance was fabricated which showed the power density of 37.5 kW kg-1. The facile fabrication approach and its excellent capacitance improvement, propose it as an efficient technique for enhancing the supercapacitance performance of the carbon-based electrodes.

Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.

PubMed

Santos, Jorge A N; Gouvea, Iuri E; Júdice, Wagner A S; Izidoro, Mario A; Alves, Fabiana M; Melo, Robson L; Juliano, Maria A; Skern, Tim; Juliano, Luiz

2009-08-25

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

PubMed

Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

2015-12-11

Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reactive ion etched substrates and methods of making and using

DOEpatents

Rucker, Victor C [San Francisco, CA; Shediac, Rene [Oakland, CA; Simmons, Blake A [San Francisco, CA; Havenstrite, Karen L [New York, NY

2007-08-07

Disclosed herein are substrates comprising reactive ion etched surfaces and specific binding agents immobilized thereon. The substrates may be used in methods and devices for assaying or isolating analytes in a sample. Also disclosed are methods of making the reactive ion etched surfaces.

Substrate Material for Holographic Emulsions Utilizing Fluorinated Polyimide Film

NASA Technical Reports Server (NTRS)

Gierow, Paul A. (Inventor); Clayton, William R. (Inventor); St.Clair, Anne K. (Inventor)

1999-01-01

A new holographic substrate utilizing flexible. optically transparent fluorinated polyimides. Said substrates have 0 extremely low birefringence which results in a high signal to noise ratio in subsequent holograms. Specific examples of said fluorinated polyimides include 6FDA+APB and 6FDA+4BDAF.

Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.

PubMed

Andersen, Rokhsana J; Brask, Jesper

2016-06-01

Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Wafer bonded virtual substrate and method for forming the same

DOEpatents

Atwater, Jr., Harry A.; Zahler, James M [Pasadena, CA; Morral, Anna Fontcuberta i [Paris, FR

2007-07-03

A method of forming a virtual substrate comprised of an optoelectronic device substrate and handle substrate comprises the steps of initiating bonding of the device substrate to the handle substrate, improving or increasing the mechanical strength of the device and handle substrates, and thinning the device substrate to leave a single-crystal film on the virtual substrate such as by exfoliation of a device film from the device substrate. The handle substrate is typically Si or other inexpensive common substrate material, while the optoelectronic device substrate is formed of more expensive and specialized electro-optic material. Using the methodology of the invention a wide variety of thin film electro-optic materials of high quality can be bonded to inexpensive substrates which serve as the mechanical support for an optoelectronic device layer fabricated in the thin film electro-optic material.

Wafer bonded virtual substrate and method for forming the same

NASA Technical Reports Server (NTRS)

Atwater, Jr., Harry A. (Inventor); Zahler, James M. (Inventor); Morral, Anna Fontcuberta i (Inventor)

2007-01-01

A method of forming a virtual substrate comprised of an optoelectronic device substrate and handle substrate comprises the steps of initiating bonding of the device substrate to the handle substrate, improving or increasing the mechanical strength of the device and handle substrates, and thinning the device substrate to leave a single-crystal film on the virtual substrate such as by exfoliation of a device film from the device substrate. The handle substrate is typically Si or other inexpensive common substrate material, while the optoelectronic device substrate is formed of more expensive and specialized electro-optic material. Using the methodology of the invention a wide variety of thin film electro-optic materials of high quality can be bonded to inexpensive substrates which serve as the mechanical support for an optoelectronic device layer fabricated in the thin film electro-optic material.

Photocathode

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

Opachich, Yekaterina; MacPhee, Andrew

A photocathode designs that leverage the grazing incidence geometry yield improvements through the introduction of recessed structures, such as cones, pyramids, pillars or cavities to the photocathode substrate surface. Improvements in yield of up to 20 times have been shown to occur in grazing incidence geometry disclosed herein due to a larger path length of the X-ray photons which better matches the secondary electron escape depth within the photocathode material. A photocathode includes a substrate having a first side and a second side, the first side configured to receive x-ray energy and the second side opposing the first side. Amore » structured surface is associated with the second side of the substrate such that the structured surface includes a plurality of recesses from the second side of the substrate into the substrate.« less

SS-mPEG chemical modification of recombinant phospholipase C for enhanced thermal stability and catalytic efficiency.

PubMed

Fang, Xian; Wang, Xueting; Li, Guiling; Zeng, Jun; Li, Jian; Liu, Jingwen

2018-05-01

PEGylation is one of the most promising and extensively studied strategies for improving the properties of proteins as well as enzymic physical and thermal stability. Phospholipase C, hydrolyzing the phospholipids offers tremendous applications in diverse fields. However, the poor thermal stability and higher cost of production have restricted its industrial application. This study focused on improving the stabilization of recombinant PLC by chemical modification with methoxypolyethylene glycol-Succinimidyl Succinate (SS-mPEG, MW 5000). PLC gene from isolate Bacillus cereus HSL3 was fused with SUMO, a novel small ubiquitin-related modifier expression vector and over expressed in Escherichia coli. The soluble fraction of SUMO-PLC reached 80% of the total recombinant protein. The enzyme exhibited maximum catalytic activity at 80 °C and was relatively thermostable at 40-70 °C. It showed extensive substrate specificity pattern and marked activity toward phosphatidylcholine, which made it a typical non-specific PLC for industrial purpose. SS-mPEG-PLC complex exhibited an enhanced thermal stability at 70-80 °C and the catalytic efficiency (K cat /K m ) had increased by 3.03 folds compared with free PLC. CD spectrum of SS-mPEG-PLC indicated a possible enzyme aggregation after chemical modification, which contributed to the higher thermostability of SS-mPEG-PLC. The increase of antiparallel β sheets in secondary structure also made it more stable than parallel β sheets. The presence of SS-mPEG chains on the enzyme molecule surface somewhat changed the binding rate of the substrates, leading to a significant improvement in catalytic efficiency. This study provided an insight into the addition of SS-mPEG for enhancing the industrial applications of phospholipase C at higher temperature. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of Proteolysis by Human Deubiquitinating Enzymes

PubMed Central

Eletr, Ziad M.; Wilkinson, Keith D.

2013-01-01

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. PMID:23845989

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.

PubMed

Fedoreyeva, L I; Vanyushin, B F

2011-06-01

Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.

Mutant fatty acid desaturase

DOEpatents

Shanklin, John; Cahoon, Edgar B.

2004-02-03

The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

An Overview of P450 Enzymes: Opportunity and Challenges in Industrial Applications

DOE PAGES

Notonier, Sandra; Alexander, Meyers; Jayakody, Lahiru N.

2016-10-23

Cytochrome P450 enzymes (P450s) containing a heme-iron center, are biocatalysts from all kingdoms, involvedin a large variety of reactions. Their potential in catalyzing a broad range of substrates makes perfect candidates for biotechnology applications and the production of high-value compounds. Biocatalytic reactions performed by P450s have a great interest in the pharmaceutical industry, fine chemicals, cosmetics, and for bioremediation procedures. However, the complex nature of this protein is still a major hurdle in the prospect of using their promising ability for expanding the number of industrial applications. Multiple approaches of protein engineering are currently conducted to improve activity, stability and/ormore » substrate specificity for a given reaction. Furthermore, in combination with the appropriate biocatalyst, a suitable bioengineering process is a key step in the implementation of P450s at the industrial scale.« less

Metal chelate process to remove pollutants from fluids

DOEpatents

Chang, Shih-Ger T.

1994-01-01

The present invention relates to improved methods using an organic iron chelate to remove pollutants from fluids, such as flue gas. Specifically, the present invention relates to a process to remove NO.sub.x and optionally SO.sub.2 from a fluid using a metal ion (Fe.sup.2+) chelate wherein the ligand is a dimercapto compound wherein the --SH groups are attached to adjacent carbon atoms (HS--C--C--SH) or (SH--C--CCSH) and contain a polar functional group so that the ligand of DMC chelate is water soluble. Alternatively, the DMC' is covalently attached to a water insoluble substrate such as a polymer or resin, e.g., polystyrene. The chelate is regenerated using electroreduction or a chemical additive. The dimercapto compound bonded to a water insoluble substrate is also useful to lower the concentration or remove hazardous metal ions from an aqueous solution.

Metal chelate process to remove pollutants from fluids

DOEpatents

Chang, S.G.T.

1994-12-06

The present invention relates to improved methods using an organic iron chelate to remove pollutants from fluids, such as flue gas. Specifically, the present invention relates to a process to remove NO[sub x] and optionally SO[sub 2] from a fluid using a metal ion (Fe[sup 2+]) chelate wherein the ligand is a dimercapto compound wherein the --SH groups are attached to adjacent carbon atoms (HS--C--C--SH) or (SH--C--CCSH) and contain a polar functional group so that the ligand of DMC chelate is water soluble. Alternatively, the DMC is covalently attached to a water insoluble substrate such as a polymer or resin, e.g., polystyrene. The chelate is regenerated using electroreduction or a chemical additive. The dimercapto compound bonded to a water insoluble substrate is also useful to lower the concentration or remove hazardous metal ions from an aqueous solution. 26 figures.

An Overview of P450 Enzymes: Opportunity and Challenges in Industrial Applications

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

Notonier, Sandra; Alexander, Meyers; Jayakody, Lahiru N.

Cytochrome P450 enzymes (P450s) containing a heme-iron center, are biocatalysts from all kingdoms, involvedin a large variety of reactions. Their potential in catalyzing a broad range of substrates makes perfect candidates for biotechnology applications and the production of high-value compounds. Biocatalytic reactions performed by P450s have a great interest in the pharmaceutical industry, fine chemicals, cosmetics, and for bioremediation procedures. However, the complex nature of this protein is still a major hurdle in the prospect of using their promising ability for expanding the number of industrial applications. Multiple approaches of protein engineering are currently conducted to improve activity, stability and/ormore » substrate specificity for a given reaction. Furthermore, in combination with the appropriate biocatalyst, a suitable bioengineering process is a key step in the implementation of P450s at the industrial scale.« less

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Release of wine monoterpenes from natural precursors by glycosidases from Oenococcus oeni

PubMed Central

Michlmayr, Herbert; Nauer, Stefan; Brandes, Walter; Schümann, Christina; Kulbe, Klaus D.; del Hierro, Andrés M.; Eder, Reinhard

2012-01-01

It is now well established that wine-related lactic acid bacteria (LAB), especially Oenococcus oeni, possess glycosidase activities that positively contribute to wine aroma through the hydrolysis of grape-derived aroma precursors. In our recent studies, we have identified and characterised several LAB glycosidases with potential in these terms. Here, we report that both a glucosidase and an arabinosidase from O. oeni can release high amounts of monoterpenes from natural substrates under optimal conditions, indicating that these intracellular enzymes might play a significant role in the hydrolysis of aroma precursors during malolactic fermentation. The enzymes from O. oeni exhibited broad substrate specificities (release of both primary/tertiary terpene alcohols) and were even active in grape juice. Further, a sensory panel clearly preferred enzyme-treated Riesling wines over the controls and affirmed that the glycosidases from O. oeni could improve the typical Riesling aroma.

Reciprocating sliding wear evaluation of a polymeric/coating tribological system

NASA Astrophysics Data System (ADS)

Braza, J. F.; Furst, R. E.

1993-04-01

Reciprocating screening tests aimed at simulating a control bearing in a contaminated environment to discern the optimum polymeric/coating combination are described. The polymeric/coating systems were compared with the wear of a baseline phenolic impregnated polytetrafluoroethylene (PTFE) polyester woven fabric composite against an uncoated stainless steel substrate. The polymeric composites under consideration include a polyamide-imide (PAI), a polybenzimidazole, and an injection-moldable PEEK. Results indicate that the system of either PEEK or PAI with an E-Ni-PTFE- or TiN-coated substrate produced the best tribological system. These two composites also exhibited a significant improvement over the baseline fabric when tested against the high-velocity oxygen-fuel thermal spray coating. To discern better the optimum polymeric composite/coating system, full-scale testing must be conducted to study system dynamics, vibrations, counterface hardness and roughness, temperature, external environment and application specific conditions.

The effect of substrate composition and storage time on urine specific gravity in dogs.

PubMed

Steinberg, E; Drobatz, K; Aronson, L

2009-10-01

The purpose of this study is to evaluate the effects of substrate composition and storage time on urine specific gravity in dogs. A descriptive cohort study of 15 dogs. The urine specific gravity of free catch urine samples was analysed during a 5-hour time period using three separate storage methods; a closed syringe, a diaper pad and non-absorbable cat litter. The urine specific gravity increased over time in all three substrates. The syringe sample had the least change from baseline and the diaper sample had the greatest change from baseline. The urine specific gravity for the litter and diaper samples had a statistically significant increase from the 1-hour to the 5-hour time point. The urine specific gravity from canine urine stored either on a diaper or in a non-absorbable litter increased over time. Although the change was found to be statistically significant over the 5-hour study period it is unlikely to be clinically significant.

Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

PubMed

MacDonald, Logan C; Berger, Bryan W

2014-06-27

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

2015-01-01

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

PubMed Central

Park, Eunsook; Woo, Jongchan; Dinesh-Kumar, SP

2014-01-01

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes. PMID:24658121

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4.

PubMed

Fang, Shujun; Chang, Jie; Lee, Yong Seok; Guo, Weiliang; Choi, Yong Lark; Zhou, Yongcan

2014-01-01

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure-function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.

Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

PubMed Central

Deu, Edgar; Yang, Zhimou; Wang, Flora; Klemba, Michael; Bogyo, Matthew

2010-01-01

Background High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic. PMID:20700487

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.

PubMed

Chen, Sheng; Wan, Hoi Ying

2011-01-15

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S-transferase from Acidovorax sp. KKS102.

PubMed

Shehu, Dayyabu; Alias, Zazali

2018-05-19

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Investigation of Low Cost Substrate Approaches for III-V Solar Cells

NASA Astrophysics Data System (ADS)

Lichty, Marlene Lydia

With the need for cleaner energy sources, which can displace fossil fuel, the solar cell industry is of particular interest due to the abundancy of the Sun. Silicon currently dominates terrestrial applications, but efficiency improvements have saturated. III-V based solar cells have reported the highest efficiencies, however, high costs due to substrates and fabrication processes have limited these devices to specialty applications, such as space. In order to reduce the cost associated with fabricating III-V semiconductor substrate material, two different approaches were taken in this work with a particular focus on making III-Vs more applicable outside of specialty applications, including InP, InAsnd Ge. Typical material characterization techniques were used to analyze the samples and processes studied in this thesis. The first process examined was the direct epitaxial growth of III-V materials by MOCVD on cheaper substrates. More specifically, the direct growth of InP and InAs on metal foils. A growth time study and surface coverage analysis was performed for the growth of InP. A characterization study was then conducted on the second process, the aluminum- induced crystallization of germanium to determine the effects this process had on the surface. Crystalline InP, InAs and Ge were successfully characterized in this work, and show promise for use in cheaper III-V alternatives to terrestrial energy solutions.

The safety of lumacaftor and ivacaftor for the treatment of cystic fibrosis.

PubMed

Talamo Guevara, Maria; McColley, Susanna A

2017-11-01

Lumacaftor-ivacaftor is indicated for treatment of cystic fibrosis (CF) in patients homozygous for the Phe-508del cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. In clinical trials, treated patients showed improved pulmonary function, reduced pulmonary exacerbations, and other benefits. This article reviews safety of this therapy. Areas covered: Safety findings in ivacaftor, lumacaftor and combined therapy trials, and reported subsequently through post-approval evaluation, were accessed by PubMed and Google searches using key words 'VX-770', 'ivacaftor', 'VX-809', and 'lumacaftor'. Transaminitis was seen in ivacaftor and combination trials. Non-congenital cataracts were seen in pre-clinical animal studies and in children taking ivacaftor and combined therapy. Dyspnea occurs in some patients taking lumacaftor and combined therapy and usually resolves without stopping treatment. Lumacaftor is a strong inducer of CYP3A while ivacaftor is a CYP3A sensitive substrate. Combination therapy can decrease systemic exposure of medications that are substrates of CYP3A, decreasing therapeutic effect. Co-administration of lumacaftor-ivacaftor with sensitive CYP3A substrates or CYP3A substrates with narrow therapeutic index is not recommended. Expert opinion: Lumacaftor-ivacaftor therapy may be associated with ocular and hepatic side effects. Specific recommendations for monitoring are available. Dyspnea occurs, especially during initiation of treatment. Potential drug interactions should be evaluated in patients taking combination therapy. The risk benefit ratio of lumacaftor-ivacaftor favors therapy.

Direct measurement of catalase activity in living cells and tissue biopsies.

PubMed

Scaglione, Christine N; Xu, Qijin; Ramanujan, V Krishnan

2016-01-29

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies - can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Influential factors on thermoacoustic efficiency of multilayered graphene film loudspeakers for optimal design

NASA Astrophysics Data System (ADS)

Xing, Qianhe; Li, Shuang; Fan, Xueliang; Bian, Anhua; Cao, Shi-Jie; Li, Cheng

2017-09-01

Graphene thermoacoustic loudspeakers, composed of a graphene film on a substrate, generate sound with heat. Improving thermoacoustic efficiency of graphene speakers is a goal for optimal design. In this work, we first modified the existing TA model with respect to small thermal wavelengths, and then built an acoustic platform for model validation. Additionally, sensitivity analyses for influential factors on thermoacoustic efficiency were performed, including the thickness of multilayered graphene films, the thermal effusivity of substrates, and the characteristics of inserted gases. The higher sensitivity coefficients result in the stronger effects on thermoacoustic efficiency. We find that the thickness (5 nm-15 nm) of graphene films plays a trivial role in efficiency, resulting in the sensitivity coefficient less than 0.02. The substrate thermal effusivity, however, has significant effects on efficiency, with the sensitivity coefficient around 1.7. Moreover, substrates with a lower thermal effusivity show better acoustic performances. For influences of ambient gases, the sensitivity coefficients of density ρg, thermal conductivity κg, and specific heat cp,g are 2.7, 0.98, and 0.8, respectively. Furthermore, large magnitudes of both ρg and κg lead to a higher efficiency and the sound pressure level generated by graphene films is approximately proportional to the inverse of cp,g. These findings can refer to the optimal design for graphene thermoacoustic speakers.

Direct Measurement of Catalase Activity in Living Cells and Tissue Biopsies

PubMed Central

Scaglione, Christine N; Xu, Qijin; Ramanujan, V. Krishnan

2016-01-01

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharamacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. PMID:26772884

Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

PubMed Central

Chen, Zhiwei; Pereira, Jose H.; Liu, Hanbin; Tran, Huu M.; Hsu, Nathan S. Y.; Dibble, Dean; Singh, Seema; Adams, Paul D.; Sapra, Rajat; Hadi, Masood Z.; Simmons, Blake A.; Sale, Kenneth L.

2013-01-01

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding. PMID:24244549

Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

PubMed

Chen, Zhiwei; Pereira, Jose H; Liu, Hanbin; Tran, Huu M; Hsu, Nathan S Y; Dibble, Dean; Singh, Seema; Adams, Paul D; Sapra, Rajat; Hadi, Masood Z; Simmons, Blake A; Sale, Kenneth L

2013-01-01

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.

Effect of tacticity on the structure and glass transition temperature of polystyrene adsorbed onto solid surfaces

NASA Astrophysics Data System (ADS)

Negash, Solomon; Tatek, Yergou B.; Tsige, Mesfin

2018-04-01

We have carried out atomistic (all-atom) molecular dynamics simulations to investigate the effect of tacticity on the structure and glass transition temperature (Tg) of polystyrene (PS) thin films adsorbed on two distinct types of solid substrates. The systems consist of thin films made of atactic, isotactic, and syndiotactic PS chains supported by graphite or hydroxylated α-quartz substrates, which are known to be atomically flat but chemically and structurally different. We have observed a marked dependence of the film structure on substrate type as well as on tacticity. For instance, rings' orientation near substrate surfaces depends on substrate type for atactic PS and isotactic PS films, while no such dependence is observed for syndiotactic PS films whose interfacial structure seems to result from their propensity to adopt the trans conformation rather than their specific interaction with the substrates. Moreover, our results indicate that glass transition temperatures of substrate supported polystyrene films are higher compared to those of the corresponding free-standing films. More specifically, PS films on graphite exhibit larger Tg values than those on α-quartz, and we have noticed that syndiotactic PS has the largest Tg irrespective of the substrate type. Furthermore, the local Tg in the region of the film in contact with the substrates shows a strong tacticity and substrate dependence, whereas no dependencies were found for the local Tg in the middle of the film. Substrate-film interaction energy and chains' dynamics near substrate-film interfaces were subsequently investigated in order to substantiate the obtained Tgs, and it was found that films with higher Tgs are strongly adsorbed on the substrates and/or exhibit smaller interfacial chains' dynamics essentially due to steric hindrance.

Sudden substrate dilution induces a higher rate of citric acid production by Aspergillus niger.

PubMed Central

Legisa, M; Gradisnik-Grapulin, M

1995-01-01

On the basis of the present knowledge of Aspergillus niger metabolism during citric acid fermentation, an idea on how to improve the process was formed. Initially, a higher sucrose concentration was used for the germination of spores, which caused a higher intracellular level of the osmoregulator, glycerol, to be present. When citric acid started to be excreted into the medium, the substrate was suddenly diluted. Optimization of this procedure resulted in a nearly tripled volumetric rate (grams per liter per hour) of acid production, while the overall fermentation time was halved compared with the usual batch process. Yet, a characteristic delay was observed at the start of the acid excretion after the dilution. Hypo-osmotic shock caused a prominent elevation of intracellular cyclic AMP levels. Simultaneously, the specific activity of 6-phosphofructo-1-kinase increased significantly, probably due to phosphorylation of the protein molecule by cyclic AMP-dependent protein kinase. Specific 6-phosphofructo-1-kinase activity was much higher in the treated than in the normally growing mycelium. The metabolic flow through glycolysis was expected to be higher, which should contribute to a higher volumetric rate of acid production. PMID:7618885

Sudden substrate dilution induces a higher rate of citric acid production by Aspergillus niger.

PubMed

Legisa, M; Gradisnik-Grapulin, M

1995-07-01

On the basis of the present knowledge of Aspergillus niger metabolism during citric acid fermentation, an idea on how to improve the process was formed. Initially, a higher sucrose concentration was used for the germination of spores, which caused a higher intracellular level of the osmoregulator, glycerol, to be present. When citric acid started to be excreted into the medium, the substrate was suddenly diluted. Optimization of this procedure resulted in a nearly tripled volumetric rate (grams per liter per hour) of acid production, while the overall fermentation time was halved compared with the usual batch process. Yet, a characteristic delay was observed at the start of the acid excretion after the dilution. Hypo-osmotic shock caused a prominent elevation of intracellular cyclic AMP levels. Simultaneously, the specific activity of 6-phosphofructo-1-kinase increased significantly, probably due to phosphorylation of the protein molecule by cyclic AMP-dependent protein kinase. Specific 6-phosphofructo-1-kinase activity was much higher in the treated than in the normally growing mycelium. The metabolic flow through glycolysis was expected to be higher, which should contribute to a higher volumetric rate of acid production.

Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

PubMed

Poon, Kar Lai; Wang, Xingang; Ng, Ashley S; Goh, Wei Huang; McGinnis, Claudia; Fowler, Stephen; Carney, Tom J; Wang, Haishan; Ingham, Phillip W

2017-03-01

Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

Arylethynyl receptors for neutral molecules and anions: emerging applications in cellular imaging.

PubMed

Carroll, Calden N; Naleway, John J; Haley, Michael M; Johnson, Darren W

2010-10-01

This critical review will focus on the application of shape-persistent receptors for anions that derive their rigidity and optoelectronic properties from the inclusion of arylethynyl linkages. It will highlight a few of the design strategies involved in engineering selective and sensitive fluorescent probes and how arylacetylenes can offer a design pathway to some of the more desirable properties of a selective sensor. Additionally, knowledge gained in the study of these receptors in organic media often leads to improved receptor design and the production of chromogenic and fluorogenic probes capable of detecting specific substrates among the multitude of ions present in biological systems. In this ocean of potential targets exists a large number of geometrically distinct anions, which present their own problems to the design of receptors with complementary binding for each preferred coordination geometry. Our interest in targeting charged substrates, specifically how previous work on receptors for cations or neutral guests can be adapted to anions, will be addressed. Additionally, we will focus on the design and development of supramolecular arylethynyl systems, their shape-persistence and fluorogenic or chromogenic optoelectronic responses to complexation. We will also examine briefly how the "chemistry in the cuvet" translates into biological media (125 references).

Specific surface to evaluate the efficiencies of milling and pretreatment of wood for enzymatic saccharification

Treesearch

Junyong Zhu; G.S. Wang; X.J. Pan; Roland Gleisner

2009-01-01

Sieving methods have been almost exclusively used for feedstock size-reduction characterization in the biomass refining literature. This study demonstrates a methodology to properly characterize specific surface of biomass substrates through two dimensional measurement of each fiber of the substrate using a wet imaging technique. The methodology provides more...

Better Back Contacts for Solar Cells on Flexible Substrates

NASA Technical Reports Server (NTRS)

Woods, Lawrence M.; Ribelin, Rosine M.

2006-01-01

Improved low-resistance, semitransparent back contacts, and a method of fabricating them, have been developed for solar photovoltaic cells that are made from thin films of I-III-VI2 semiconductor materials on flexible, high-temperatureresistant polyimide substrates or superstrates. The innovative aspect of the present development lies in the extension, to polyimide substrates or superstrates, of a similar prior development of improved low-resistance, semitransparent back contacts for I-III-VI2 solar cells on glass substrates or superstrates. A cell incorporating this innovation can be used either as a stand-alone photovoltaic device or as part of a monolithic stack containing another photovoltaic device that utilizes light of longer wavelengths.

Phospholipid substrate-specificity of the L-serine base-exchange enzyme in rat liver microsomal fraction.

PubMed Central

Bjerve, K S

1984-01-01

The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines. PMID:6430274

Surface Effects and Challenges for Application of Piezoelectric Langasite Substrates in Surface Acoustic Wave Devices Caused by High Temperature Annealing under High Vacuum.

PubMed

Seifert, Marietta; Rane, Gayatri K; Kirbus, Benjamin; Menzel, Siegfried B; Gemming, Thomas

2015-12-19

Substrate materials that are high-temperature stable are essential for sensor devices which are applied at high temperatures. Although langasite is suggested as such a material, severe O and Ga diffusion into an O-affine deposited film was observed during annealing at high temperatures under vacuum conditions, leading to a damage of the metallization as well as a change of the properties of the substrate and finally to a failure of the device. Therefore, annealing of bare LGS (La 3 Ga 5 SiO 14 ) substrates at 800 ∘ C under high vacuum conditions is performed to analyze whether this pretreatment improves the suitability and stability of this material for high temperature applications in vacuum. To reveal the influence of the pretreatment on the subsequently deposited metallization, RuAl thin films are used as they are known to oxidize on LGS at high temperatures. A local study of the pretreated and metallized substrates using transmission electron microscopy reveals strong modification of the substrate surface. Micro cracks are visible. The composition of the substrate is strongly altered at those regions. Severe challenges for the application of LGS substrates under high-temperature vacuum conditions arise from these substrate damages, revealing that the pretreatment does not improve the applicability.

Synthesis of Platinum-nickel Nanowires and Optimization for Oxygen Reduction Performance.

PubMed

Alia, Shaun M; Pivovar, Bryan S

2018-04-27

Platinum-nickel (Pt-Ni) nanowires were developed as fuel cell electrocatalysts, and were optimized for the performance and durability in the oxygen reduction reaction. Spontaneous galvanic displacement was used to deposit Pt layers onto Ni nanowire substrates. The synthesis approach produced catalysts with high specific activities and high Pt surface areas. Hydrogen annealing improved Pt and Ni mixing and specific activity. Acid leaching was used to preferentially remove Ni near the nanowire surface, and oxygen annealing was used to stabilize near-surface Ni, improving durability and minimizing Ni dissolution. These protocols detail the optimization of each post-synthesis processing step, including hydrogen annealing to 250 °C, exposure to 0.1 M nitric acid, and oxygen annealing to 175 °C. Through these steps, Pt-Ni nanowires produced increased activities more than an order of magnitude than Pt nanoparticles, while offering significant durability improvements. The presented protocols are based on Pt-Ni systems in the development of fuel cell catalysts. These techniques have also been used for a variety of metal combinations, and can be applied to develop catalysts for a number of electrochemical processes.

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.

PubMed

Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio

2011-04-01

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

PubMed

Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof

2011-01-01

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.

PubMed

Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence

2016-03-08

Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Different substrates and starter inocula govern microbial community structures in biogas reactors.

PubMed

Satpathy, Preseela; Steinigeweg, Sven; Cypionka, Heribert; Engelen, Bert

2016-01-01

The influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.

Performance improvements of binary diffractive structures via optimization of the photolithography and dry etch processes

NASA Astrophysics Data System (ADS)

Welch, Kevin; Leonard, Jerry; Jones, Richard D.

2010-08-01

Increasingly stringent requirements on the performance of diffractive optical elements (DOEs) used in wafer scanner illumination systems are driving continuous improvements in their associated manufacturing processes. Specifically, these processes are designed to improve the output pattern uniformity of off-axis illumination systems to minimize degradation in the ultimate imaging performance of a lithographic tool. In this paper, we discuss performance improvements in both photolithographic patterning and RIE etching of fused silica diffractive optical structures. In summary, optimized photolithographic processes were developed to increase critical dimension uniformity and featuresize linearity across the substrate. The photoresist film thickness was also optimized for integration with an improved etch process. This etch process was itself optimized for pattern transfer fidelity, sidewall profile (wall angle, trench bottom flatness), and across-wafer etch depth uniformity. Improvements observed with these processes on idealized test structures (for ease of analysis) led to their implementation in product flows, with comparable increases in performance and yield on customer designs.

Calcium phosphate coatings on magnesium alloys for biomedical applications: a review.

PubMed

Shadanbaz, Shaylin; Dias, George J

2012-01-01

Magnesium has been suggested as a revolutionary biodegradable metal for use as an orthopaedic material. As a biocompatible and degradable metal, it has several advantages over the permanent metallic materials currently in use, including eliminating the effects of stress shielding, improving biocompatibility concerns in vivo and improving degradation properties, removing the requirement of a second surgery for implant removal. The rapid degradation of magnesium, however, is a double-edged sword as it is necessary to control the corrosion rates of the materials to match the rates of bone healing. In response, calcium phosphate coatings have been suggested as a means to control these corrosion rates. The potential calcium phosphate phases and their coating techniques on substrates are numerous and can provide several different properties for different applications. The reactivity and low melting point of magnesium, however, require specific parameters for calcium phosphate coatings to be successful. Within this review, an overview of the different calcium phosphate phases, their properties and their behaviour in vitro and in vivo has been provided, followed by the current coating techniques used for calcium phosphates that may be or may have been adapted for magnesium substrates. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Applications of fibrous substrates containing insolubilized phase change polymers

NASA Technical Reports Server (NTRS)

Vigo, Tyrone L.; Bruno, Joseph S.

1993-01-01

Incorporation of polyethylene glycols into fibrous substrates produces several improved functional properties when they are insolubilized by crosslinking with a methylolamide resin or by polyacetal formation by their reaction with glyoxal. The range of molecular weights of polyols that may be insolubilized is broad as are the curing conditions (0.25-10 min at 80-200C). Most representative fiber types and blends (natural and synthetic) and all types of fabric constructions (woven, nonwoven and knit) have been modified by incorporation of the bound polyols. The most novel property is the thermal adaptability of the modified substrates to many climatic conditions. This adaptability is due to the high latent heat of the crosslinked polyols that function as phase change materials, the hydrophilic nature of the crosslinked polymer and its enhanced thermal conductivity. Other enhanced properties imparted to fabrics include flex and flat abrasion, antimicrobial activity, reduced static charge, resistance to oily soils, resiliency, wind resistance and reduced lint loss. Applications commercialized in the U.S. and Japan include sportswear and skiwear. Several examples of electric sets of properties useful for specific end uses are given. In addition, other uses are biomedical horticultural, aerospace, indoor insulation, automotive interiors and components and packaging material.

Durable anti-fogging effect and adhesion improvement on polymer surfaces

NASA Astrophysics Data System (ADS)

Moser, E. M.; Gilliéron, D.; Henrion, G.

2010-01-01

The hydrophobic properties of polymeric surfaces may cause fogging in transparent packaging and poor adhesion to printing colours and coatings. Novel plasma processes for durable functionalization of polypropylene and polyethylene terephthalate substrates were developed and analysed using optical emission spectroscopy. A worm-like nano pattern was created on the polypropylene surface prior to the deposition of thin polar plasma polymerised layers. For both substrates, highly polar surfaces exhibiting a surface tension of up to 69 mN/m and a water contact angle of about 10° were produced - providing the anti-fogging effect. The deposition of thin plasma polymerised layers protects the increased surface areas and enables to tailoring the surface energy of the substrate in a wide range. Wetting characteristics were determined by dynamic contact angle measurements. Investigations of the chemical composition of several layers using X-ray photoelectron spectroscopy and FT-infrared spectroscopy were correlated with functional testing. The surface topography was investigated using atomic force microscopy. The weldability and peeling-off characteristics of the plasma treated polymer films could be adjusted by varying the process parameters. Global and specific migration analyses were undertaken in order to ensure the manufacturing of plasma treated polymer surfaces for direct food contact purposes.

Biofuel cell operating on activated THP-1 cells: A fuel and substrate study.

PubMed

Javor, Kristina; Tisserant, Jean-Nicolas; Stemmer, Andreas

2017-01-15

It is known that electrochemical energy can be harvested from mammalian cells, more specifically from white blood cells (WBC). This study focuses on an improved biofuel cell operating on phorbol myristate acetate (PMA) activated THP-1 human monocytic cells. Electrochemical investigation showed strong evidence pointing towards hydrogen peroxide being the primary current source, confirming that the current originates from NADPH oxidase activity. Moreover, an adequate substrate for differentiation and activation of THP-1 cells was examined. ITO, gold, platinum and glass were tested and the amount of superoxide anion produced by NADPH oxidase was measured by spectrophotometry through WST-1 reduction at 450nm and used as an indicator of cellular activity and viability. These substrates were subsequently used in a conventional two-compartment biofuel cell where the power density output was recorded. The material showing the highest cell activity compared to the reference cell culture plate and the highest power output was ITO. Under our experimental conditions, a power density of 4.5μW/cm 2 was reached. To the best of our knowledge, this is a threefold higher power output than other leukocyte biofuel cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Applications of fibrous substrates containing insolubilized phase change polymers

NASA Astrophysics Data System (ADS)

Vigo, Tyrone L.; Bruno, Joseph S.

1993-02-01

Incorporation of polyethylene glycols into fibrous substrates produces several improved functional properties when they are insolubilized by crosslinking with a methylolamide resin or by polyacetal formation by their reaction with glyoxal. The range of molecular weights of polyols that may be insolubilized is broad as are the curing conditions (0.25-10 min at 80-200C). Most representative fiber types and blends (natural and synthetic) and all types of fabric constructions (woven, nonwoven and knit) have been modified by incorporation of the bound polyols. The most novel property is the thermal adaptability of the modified substrates to many climatic conditions. This adaptability is due to the high latent heat of the crosslinked polyols that function as phase change materials, the hydrophilic nature of the crosslinked polymer and its enhanced thermal conductivity. Other enhanced properties imparted to fabrics include flex and flat abrasion, antimicrobial activity, reduced static charge, resistance to oily soils, resiliency, wind resistance and reduced lint loss. Applications commercialized in the U.S. and Japan include sportswear and skiwear. Several examples of electric sets of properties useful for specific end uses are given. In addition, other uses are biomedical horticultural, aerospace, indoor insulation, automotive interiors and components and packaging material.

Batch production of Pyranose 2-oxidase from Trametes versicolor (ATCC 11235) in medium with a lignocellulosic substrate and enzymatic bleaching of cotton fabrics.

PubMed

Pazarlioglu, Nurdan Kasikara; Erden, Emre; Ucar, M Cigdem; Akkaya, Alper; Sariisik, A Merih

2012-04-01

The aim of this work was to determine new, different and low-cost substrates that can be used for enzyme production from the white rot fungus Trametes versicolor (ATCC 11235) by taking advantage of the broad substrate specificity of pyranose 2-oxidase. In this report, we investigated the production of pyranose 2-oxidase from T. versicolor (ATCC 11235) using ten different agricultural residues such as clover straw, almond shells, hazelnut cobs, grass and others. Pyranose 2-oxidase activity was determined as 2.332 U/g at the 9th day in a submerged culture containing clover straw and tap water shaken at 150 rpm and 26°C, and the optimum clover straw concentration was determined to be 12 g/l. The effects of different glucose, nitrogen and phosphate sources on the production of pyranose 2-oxidase were studied in the clover straw medium. Analyses of biomass, protein, reduced sugar and nitrogen concentrations were also monitored in a clover straw medium that did not contain carbon or nitrogen and phosphate sources under the parameters determined. The produced pyranose 2-oxidase was used for improving the properties of cotton fabrics.

Photochemical synthesis of the Fe0/C3N4/MoS2 heterostructure as a highly active and reusable photocatalyst

NASA Astrophysics Data System (ADS)

Wang, Xiu; Zhou, Zhiming; Liang, Zhiyu; Zhuang, Zanyong; Yu, Yan

2017-11-01

The Fe0/C3N4/MoS2 heterostructure was fabricated through photochemical synthesis that was free of NaBH4. Specifically, the g-C3N4/MoS2 (GCNM) composite was used as the substrate. Visible light excited the electrons from the valence band of the GCNM in the substrate, and the excited electrons reduced the Fe2+ ions in the solution nearby GCNM to Fe0 and then created the Fe0/C3N4/MoS2 heterostructure. Small Fe0 (<9 nm) dots well dispersed on the GCNM surface were obtained, because the diffusion of the Fe ions in the solution and the diffusion of the electrons on the GCNM substrate restricted the growth of Fe0 nanoparticles. The smaller size of Fe0 provided a larger number of active metal centers and improved the carrier separation efficiency. As a result, the Fe0/C3N4/MoS2 heterostructure exhibited superior catalytic properties in the redox reactions of rhodamine B, Cr(VI), Pb(II), and Cd(II). It could also be readily recycled without severe loss of catalytic performance.

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

PubMed Central

Klaus, Maja; Ostrowski, Matthew P.; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E.; Khosla, Chaitan

2016-01-01

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns

USGS Publications Warehouse

Haack, S.K.; Garchow, H.; Klug, M.J.; Forney, L.J.

1995-01-01

We determined factors that affect responses of bacterial isolates and model bacterial communities to the 95 carbon substrates in Biolog microliter plates. For isolates and communities of three to six bacterial strains, substrate oxidation rates were typically nonlinear and were delayed by dilution of the inoculum. When inoculum density was controlled, patterns of positive and negative responses exhibited by microbial communities to each of the carbon sources were reproducible. Rates and extents of substrate oxidation by the communities were also reproducible but were not simply the sum of those exhibited by community members when tested separately. Replicates of the same model community clustered when analyzed by principal- components analysis (PCA), and model communities with different compositions were clearly separated un the first PCA axis, which accounted for >60% of the dataset variation. PCA discrimination among different model communities depended on the extent to which specific substrates were oxidized. However, the substrates interpreted by PCA to be most significant in distinguishing the communities changed with reading time, reflecting the nonlinearity of substrate oxidation rates. Although whole-community substrate utilization profiles were reproducible signatures for a given community, the extent of oxidation of specific substrates and the numbers or activities of microorganisms using those substrates in a given community were not correlated. Replicate soil samples varied significantly in the rate and extent of oxidation of seven tested substrates, suggesting microscale heterogeneity in composition of the soil microbial community.

Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli

PubMed Central

Zimanyi, Christina M; Chen, Percival Yang-Ting; Kang, Gyunghoon; Funk, Michael A; Drennan, Catherine L

2016-01-01

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell. DOI: http://dx.doi.org/10.7554/eLife.07141.001 PMID:26754917

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466

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

Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less

Substrate specificity and copper loading of the manganese-oxidizing multicopper oxidase Mnx from Bacillus sp. PL-12.

PubMed

Butterfield, Cristina N; Tebo, Bradley M

2017-02-22

Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of k cat , but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

PubMed Central

Malyavantham, Kishore S.; Suresh, Lakshmanan

2018-01-01

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern. PMID:29364249

alpha-1,4-Glucan lyase, a new class of starch/glycogen degrading enzyme. III. Substrate specificity, mode of action, and cleavage mechanism.

PubMed

Yu, S; Ahmad, T; Kenne, L; Pedersén, M

1995-05-11

The alpha-1,4-glucan lyase (EC 4.2.2.-), purified from the red alga Gracilariopsis lemaneiformis, is a single polypeptide with a molecular mass of 116,654 Da as determined by matrix-assisted laser-desorption mass spectrometry. It degraded maltose, maltosaccharides, amylose, amylopectin and glycogen, forming 1,5-anhydro-D-fructose from the non-reducing end groups. The substrate specificity, mode of action, and cleavage mechanism of the enzyme were studied by using various naturally occurring and synthesized substrates. This enzyme was highly specific for the alpha-1,4-D-glucosidic bond. When a linear alpha-1,4-glucan was used as substrate, the enzyme split the substrate from the non-reducing end and released 1,5-anhydro-D-fructose successively until only one glucose unit was left. When a branched pentasaccharide of 6(2)-alpha-maltosylmaltotriose, obtained from glycogen by alpha-amylase limitation, was used as substrate, the glucose group in the 4-position of the 4,6-branched residue was not cleaved off. Using maltoheptaose as substrate and following the reaction with HPLC and 1H-NMR spectroscopy, it was found that the action mode of the lyase followed a multichain attack mechanism. 1H- and 13C-NMR spectroscopic studies on unlabelled and labelled amylose (1-2H, 2-2H, 1-13C) as substrates indicated that the lyase cleaved the C-(1')-O(4) bond forming a double bond between C-1' and C-2', thus forming the enol form of 1,5-anhydro-D-fructose. It also indicated that the catalytic process of the lyase involved proton exchanges among C-1, C-2, C-3 and the solvent.

Preparation and Characterization of Thin-Film Composite Membrane with Nanowire-Modified Support for Forward Osmosis Process

PubMed Central

Low, Ze-Xian; Liu, Qi; Shamsaei, Ezzatollah; Zhang, Xiwang; Wang, Huanting

2015-01-01

Internal concentration polarization (ICP) in forward osmosis (FO) process is a characteristic problem for asymmetric thin-film composite (TFC) FO membrane which leads to lower water flux. To mitigate the ICP effect, modification of the substrates’ properties has been one of the most effective methods. A new polyethersulfone-based ultrafiltration membrane with increased surface porosity and high water flux was recently produced by incorporating Zn2GeO4 nanowires. The composite membrane was used as a substrate for the fabrication of TFC FO membrane, by coating a thin layer of polyamide on top of the substrate. The substrate and the nanowires were characterized by a range of techniques such as SEM, XRD, and contact angle goniometry. The water permeability and molecular weight cut-offs (MWCO) of the substrate; and the FO performance of the TFC membrane were also determined. The Zn2GeO4-modified membrane showed ~45% increase in water permeability and NaCl salt rejection of 80% under RO mode. In FO mode, the ratio of water flux to reverse solute flux was also improved. However, lower FO flux was obtained which could be due to ICP. The result shows that Zn2GO4 nanowire may be used as a modifier to the substrate to improve the quality of the polyamide layer on the substrate to improve the flux and selectivity, but not as effective in reducing ICP. This work demonstrates that the incorporation of nanomaterials to the membrane substrate may be an alternative approach to improve the formation of polyamide skin layer to achieve better FO performance. PMID:25803239

Human oesophagus: a convenient antigenic substrate for the determination of anti-endomysium antibodies in the serological diagnosis of coeliac disease.

PubMed

Uibo, O; Lambrechts, A; Mascart-Lemone, F

1995-01-01

Immunoglobulin (Ig) A-class anti-endomysium antibodies are superior to other current antibody tests for detecting coeliac disease. We aimed to evaluate the suitability of human oesophagus for the determination of anti-endomysium antibodies. The specificity of monkey and human oesophageal tissue as antigenic substrate were compared using indirect immunofluorescence analysis. Overall, 159 individuals were studied: 56 patients with biopsy-proven coeliac disease (39 with active disease) and 103 controls. The patients' IgA-class anti-endomysium antibodies were compared using unfixed cryostat sections of human and monkey oesophagus. Indirect immunofluorescence analysis was performed with an initial serum sample dilution of 1:5, and if positive, the highest dilution yielding a positive reaction was reported. The anti-endomysium antibody test was positive in 38 out of 39 patients with active coeliac disease using monkey oesophagus (sensitivity 97%) and in all 39 patients with active coeliac disease using human oesophagus (sensitivity 100%). Ten out of 17 coeliac patients on a gluten-free diet had positive anti-endomysium antibodies using monkey oesophagus and 12 using human oesophagus as the antigenic substrate. This test was negative in all 103 controls using both substrates. Our study shows that human oesophageal tissue can be used instead of monkey tissue for determining anti-endomysium antibodies. Human tissue is a more sensitive antigenic substrate than monkey oesophagus and can be used to determine low titres of antibodies. Improving the diagnostic sensitivity of the anti-endomysium antibody test would make an important contribution to screening for coeliac disease.

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae.

PubMed

Matzel, Philipp; Krautschick, Lukas; Höhne, Matthias

2017-10-18

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C 4 -C 8 ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cold Gas-Sprayed Deposition of Metallic Coatings onto Ceramic Substrates Using Laser Surface Texturing Pre-treatment

NASA Astrophysics Data System (ADS)

Kromer, R.; Danlos, Y.; Costil, S.

2018-04-01

Cold spraying enables a variety of metals dense coatings onto metal surfaces. Supersonic gas jet accelerates particles which undergo with the substrate plastic deformation. Different bonding mechanisms can be created depending on the materials. The particle-substrate contact time, contact temperature and contact area upon impact are the parameters influencing physicochemical and mechanical bonds. The resultant bonding arose from plastic deformation of the particle and substrate and temperature increasing at the interface. The objective was to create specific topography to enable metallic particle adhesion onto ceramic substrates. Ceramic did not demonstrate deformation during the impact which minimized the intimate bonds. Laser surface texturing was hence used as prior surface treatment to create specific topography and to enable mechanical anchoring. Particle compressive states were necessary to build up coating. The coating deposition efficiency and adhesion strength were evaluated. Textured surface is required to obtain strong adhesion of metallic coatings onto ceramic substrates. Consequently, cold spray coating parameters depend on the target material and a methodology was established with particle parameters (diameters, velocities, temperatures) and particle/substrate properties to adapt the surface topography. Laser surface texturing is a promising tool to increase the cold spraying applications.

Optically initiated silicon carbide high voltage switch

DOEpatents

Caporaso, George J [Livermore, CA; Sampayan, Stephen E [Manteca, CA; Sullivan, James S [Livermore, CA; Sanders,; David, M [Livermore, CA

2011-02-22

An improved photoconductive switch having a SiC or other wide band gap substrate material, such as GaAs and field-grading liners composed of preferably SiN formed on the substrate adjacent the electrode perimeters or adjacent the substrate perimeters for grading the electric fields.

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

1998-01-06

Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Cofactor-dependent specificity of a DEAD-box protein.

PubMed

Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin

2013-07-16

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

Proton irradiation of beryllium deposits on different candidate materials to be used as a neutron production target for accelerator-based BNCT

NASA Astrophysics Data System (ADS)

Gagetti, Leonardo; Anzorena, Manuel Suarez; Bertolo, Alma; del Grosso, Mariela; Kreiner, Andrés J.

2017-12-01

Thin Be targets for neutron production through Be(d,n) are produced and characterized. We improved and characterized the substrate surface, specifically the roughness, in order to achieve homogeneous and stable deposits. Once well bonded deposits were obtained, some of them were irradiated with a 150 keV proton beam and with a 1.45 MeV deuteron beam. Both deposits, pristine and irradiated, were characterized by profilometry, X-ray diffraction, scanning electron microscopy and electron probe microanalyzer.

Design of a prototype flow microreactor for synthetic biology in vitro.

PubMed

Boehm, Christian R; Freemont, Paul S; Ces, Oscar

2013-09-07

As a reference platform for in vitro synthetic biology, we have developed a prototype flow microreactor for enzymatic biosynthesis. We report the design, implementation, and computer-aided optimisation of a three-step model pathway within a microfluidic reactor. A packed bed format was shown to be optimal for enzyme compartmentalisation after experimental evaluation of several approaches. The specific substrate conversion efficiency could significantly be improved by an optimised parameter set obtained by computational modelling. Our microreactor design provides a platform to explore new in vitro synthetic biology solutions for industrial biosynthesis.

Broadband infrared beam splitter for spaceborne interferometric infrared sounder.

PubMed

Yu, Tianyan; Liu, Dingquan; Qin, Yang

2014-10-01

A broadband infrared beam splitter (BS) on ZnSe substrate used for the spaceborne interferometric infrared sounder (SIIRS) is studied in the spectral range of 4.44-15 μm. Both broadband antireflection coating and broadband beam-splitter coating in this BS are designed and tested. To optimize the optical properties and the stability of the BS, suitable infrared materials were selected, and improved deposition techniques were applied. The designed structures matched experimental data well, and the properties of the BS met the application specification of SIIRS.

"Prostatic acid phosphatase?" A comparison of acid phosphatase activities in epithelial cells, granulocytes, monocytes, lymphocytes, and platelets purified by velocity sedimentation in isokinetic gradients of Ficoll in tissue culture medium.

PubMed Central

Helms, S. R.; Brattain, M. G.; Pretlow, T. G.; Kreisberg, J. I.

1977-01-01

Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells. PMID:560800

High Quantum Efficiency OLED Lighting Systems

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

Shiang, Joseph

The overall goal of the program was to apply improvements in light outcoupling technology to a practical large area plastic luminaire, and thus enable the product vision of an extremely thin form factor high efficiency large area light source. The target substrate was plastic and the baseline device was operating at 35 LPW at the start of the program. The target LPW of the program was a >2x improvement in the LPW efficacy and the overall amount of light to be delivered was relatively high 900 lumens. Despite the extremely difficult challenges associated with scaling up a wet solution processmore » on plastic substrates, the program was able to make substantial progress. A small molecule wet solution process was successfully implemented on plastic substrates with almost no loss in efficiency in transitioning from the laboratory scale glass to large area plastic substrates. By transitioning to a small molecule based process, the LPW entitlement increased from 35 LPW to 60 LPW. A further 10% improvement in outcoupling efficiency was demonstrated via the use of a highly reflecting cathode, which reduced absorptive loss in the OLED device. The calculated potential improvement in some cases is even larger, ~30%, and thus there is considerable room for optimism in improving the net light coupling efficacy, provided absorptive loss mechanisms are eliminated. Further improvements are possible if scattering schemes such as the silver nanowire based hard coat structure are fully developed. The wet coating processes were successfully scaled to large area plastic substrate and resulted in the construction of a 900 lumens luminaire device.« less

Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development.

PubMed

Stribny, Jiri; Romagnoli, Gabriele; Pérez-Torrado, Roberto; Daran, Jean-Marc; Querol, Amparo

2016-03-12

The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae. In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p. The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The knowledge on the important enzyme involved in higher alcohols biosynthesis by S. kudriavzevii could be of scientific as well as of applied interest.

Prevention of corrosion with polyaniline

NASA Technical Reports Server (NTRS)

Ahmad, Naseer (Inventor); MacDiarmid, Alan G. (Inventor)

1997-01-01

Methods for improving the corrosion inhibition of a metal or metal alloy substrate surface are provided wherein the substrate surface is coated with a polyaniline film. The polyaniline film coating is applied by contacting the substrate surface with a solution of polyaniline. The polyaniline is dissolved in an appropriate organic solvent and the solvent is allowed to evaporate from the substrate surface yielding the polyaniline film coating.

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

PubMed Central

Sideris, Dionisia P.; Petrakis, Nikos; Katrakili, Nitsa; Mikropoulou, Despina; Gallo, Angelo; Ciofi-Baffoni, Simone; Banci, Lucia; Bertini, Ivano

2009-01-01

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. PMID:20026652

Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

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

Agarwal, R.; Schmidt, J; Stafford, R

2009-01-01

Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exositesmore » away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.« less

Substrate Specificity of Human Protein Arginine Methyltransferase 7 (PRMT7)

PubMed Central

Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G.

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-NG-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. PMID:25294873

Wafer bonded epitaxial templates for silicon heterostructures

DOEpatents

Atwater, Jr., Harry A.; Zahler, James M [Pasadena, CA; Morral, Anna Fontcubera I [Paris, FR

2008-03-11

A heterostructure device layer is epitaxially grown on a virtual substrate, such as an InP/InGaAs/InP double heterostructure. A device substrate and a handle substrate form the virtual substrate. The device substrate is bonded to the handle substrate and is composed of a material suitable for fabrication of optoelectronic devices. The handle substrate is composed of a material suitable for providing mechanical support. The mechanical strength of the device and handle substrates is improved and the device substrate is thinned to leave a single-crystal film on the virtual substrate such as by exfoliation of a device film from the device substrate. An upper portion of the device film exfoliated from the device substrate is removed to provide a smoother and less defect prone surface for an optoelectronic device. A heterostructure is epitaxially grown on the smoothed surface in which an optoelectronic device may be fabricated.

Wafer bonded epitaxial templates for silicon heterostructures

NASA Technical Reports Server (NTRS)

Atwater, Harry A., Jr. (Inventor); Zahler, James M. (Inventor); Morral, Anna Fontcubera I (Inventor)

2008-01-01

A heterostructure device layer is epitaxially grown on a virtual substrate, such as an InP/InGaAs/InP double heterostructure. A device substrate and a handle substrate form the virtual substrate. The device substrate is bonded to the handle substrate and is composed of a material suitable for fabrication of optoelectronic devices. The handle substrate is composed of a material suitable for providing mechanical support. The mechanical strength of the device and handle substrates is improved and the device substrate is thinned to leave a single-crystal film on the virtual substrate such as by exfoliation of a device film from the device substrate. An upper portion of the device film exfoliated from the device substrate is removed to provide a smoother and less defect prone surface for an optoelectronic device. A heterostructure is epitaxially grown on the smoothed surface in which an optoelectronic device may be fabricated.

Pd/Ni-WO3 anodic double layer gasochromic device

DOEpatents

Lee, Se-Hee; Tracy, C. Edwin; Pitts, J. Roland; Liu, Ping

2004-04-20

An anodic double layer gasochromic sensor structure for optical detection of hydrogen in improved response time and with improved optical absorption real time constants, comprising: a glass substrate; a tungsten-doped nickel oxide layer coated on the glass substrate; and a palladium layer coated on the tungsten-doped nickel oxide layer.

Improvement of carbon nanotube field emission properties by ultrasonic nanowelding

NASA Astrophysics Data System (ADS)

Zhao, Bo; Yadian, Boluo; Chen, Da; Xu, Dong; Zhang, Yafei

2008-12-01

Ultrasonic nanowelding was used to improve the field emission properties of carbon nanotube (CNT) cathodes. The CNTs were deposited on the Ti-coated glass substrate by electrophoretic deposition. By pressing CNTs against metal (Ti) substrate under a vibrating force at ultrasonic frequency, a reliable and low resistance contact was obtained between CNTs and Ti. The scanning electron microscopy results show that CNTs are embedded into the metal substrate and act as stable field emitters. The welded cathode demonstrates an excellent field emission with high emission current density and good current stability.

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Metabolic Engineering of Saccharomyces cerevisiae

PubMed Central

Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

2000-01-01

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production. PMID:10704473

Applications of Microbial Cell Sensors

NASA Astrophysics Data System (ADS)

Shimomura-Shimizu, Mifumi; Karube, Isao

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of microbial cell sensors have been developed as analytical tools. The microbial cell sensor utilizes microbes as a sensing element and a transducer. The characteristics of microbial cell sensors as sensing devices are a complete contrast to those of enzyme sensors or immunosensors, which are highly specific for the substrates of interest, although the specificity of the microbial cell sensor has been improved by genetic modification of the microbe used as the sensing element. Microbial cell sensors have the advantages of tolerance to measuring conditions, a long lifetime, and good cost performance, and have the disadvantage of a long response time. In this review, applications of microbial cell sensors are summarized.

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates*

PubMed Central

Meekins, David A.; Raththagala, Madushi; Auger, Kyle D.; Turner, Benjamin D.; Santelia, Diana; Kötting, Oliver; Gentry, Matthew S.; Vander Kooi, Craig W.

2015-01-01

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates. PMID:26231210

Highly sensitive surface-enhanced Raman scattering substrate made from superaligned carbon nanotubes.

PubMed

Sun, Yinghui; Liu, Kai; Miao, Jiao; Wang, Zheyao; Tian, Baozhong; Zhang, Lina; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2010-05-12

Surface-enhanced Raman scattering (SERS) has attracted wide attention because it can enhance normally weak Raman signal by several orders of magnitude and facilitate the sensitive detection of molecules. Conventional SERS substrates are constructed by placing metal nanoparticles on a planar surface. Here we show that, if the planar surface was substituted by a unique nanoporous surface, the enhancement effect can be dramatically improved. The nanoporous surface can be easily fabricated in batches and at low costs by cross stacking superaligned carbon nanotube films. The as-prepared transparent and freestanding SERS substrate is capable of detecting ambient trinitrotoluene vapor, showing much higher Raman enhancement than ordinary planar substrates because of the extremely large surface area and the unique zero-dimensional at one-dimensional nanostructure. These results not only provide a new approach to ultrasensitive SERS substrates, but also are helpful for improving the fundamental understanding of SERS phenomena.

Multifunctional zirconium nitride/copper multilayer coatings on medical grade 316L SS and titanium substrates for biomedical applications.

PubMed

Kumar, D Dinesh; Kaliaraj, Gobi Saravanan

2018-01-01

Protecting from wear and corrosion of many medical devices in the biomedical field is an existing scientific challenge. Surface modification with multilayer ZrN/Cu coating was deposited on medical grade stainless steel (SS) and titanium substrates to enhance their surface properties. Structural results revealed that the ZrN/Cu coatings are highly crystalline and uniform microstructure on both the substrates. Dry and wet tribological measurements of the coated titanium substrate exhibit enhanced wear resistance and low friction coefficient due to the improved microstructure. Similarly, the corrosion resistance was exceptionally improved on titanium substrates, resulting from the high inertness of coating to the SBF electrolyte solution. Antibacterial activity and epifluorescence results signify the effective killing of pathogens by means of ion release killing as well as contact killing mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improving the properties of zinc oxide thin-film surface acoustic wave device on glass substrate by introducing double alumina layers

NASA Astrophysics Data System (ADS)

Shih, Wen-Ching; Huang, Yi-Fan; Wu, Mu-Shiang

2017-10-01

ZnO films with c-axis (0002) orientation have been successfully grown by RF magnetron sputtering on Al2O3/glass substrates. The alumina films were firstly deposited on glass substrates, and then secondly deposited on interdigital transducer/ZnO film/alumina film/glass substrates by electron beam evaporation. The crystalline structure and surface roughness of the films were investigated by X-ray diffraction and atomic force microscopy, respectively. The phase velocity and coupling coefficient of the surface acoustic wave (SAW) device were both increased when we deposited the double alumina layers. On the other hand, the temperature coefficient of frequency becomes better if we increase the thickness of the lower alumina film. The experimental result is beneficial for improving the performance of the ZnO thin-film SAW devices on inexpensive glass substrates.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

1998-01-06

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

Coatings on reflective mask substrates

DOEpatents

Tong, William Man-Wai; Taylor, John S.; Hector, Scott D.; Mangat, Pawitter J. S.; Stivers, Alan R.; Kofron, Patrick G.; Thompson, Matthew A.

2002-01-01

A process for creating a mask substrate involving depositing: 1) a coating on one or both sides of a low thermal expansion material EUVL mask substrate to improve defect inspection, surface finishing, and defect levels; and 2) a high dielectric coating, on the backside to facilitate electrostatic chucking and to correct for any bowing caused by the stress imbalance imparted by either other deposited coatings or the multilayer coating of the mask substrate. An film, such as TaSi, may be deposited on the front side and/or back of the low thermal expansion material before the material coating to balance the stress. The low thermal expansion material with a silicon overlayer and a silicon and/or other conductive underlayer enables improved defect inspection and stress balancing.

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

PubMed

Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

2006-05-19

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

TMG-chitotriomycin as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases.

PubMed

Shiota, Hiroto; Kanzaki, Hiroshi; Hatanaka, Tadashi; Nitoda, Teruhiko

2013-06-28

TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was examined as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases (HexNAcases). According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were divided into 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated for their substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides but not N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligosaccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as a molecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processing HexNAcases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum.

PubMed

Stratton, Christopher F; Namanja-Magliano, Hilda A; Cameron, Scott A; Schramm, Vern L

2015-10-16

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

PubMed

Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

2015-09-01

Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

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

Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta

Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

DOE PAGES

Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; ...

2015-08-05

Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.

PubMed

Kumar, Ish; Pratt, R F

2005-08-02

The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.

Crystal Structures of the Novel Cytosolic 5′-Nucleotidase IIIB Explain Its Preference for m7GMP

PubMed Central

Monecke, Thomas; Buschmann, Juliane; Neumann, Piotr; Wahle, Elmar; Ficner, Ralf

2014-01-01

5′-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of diseases. The seven human 5′-nucleotidases differ with respect to substrate specificity and cellular localization. Recently, the novel cytosolic 5′-nucleotidase III-like protein, or cN-IIIB, has been characterized in human and Drosophila. cN-IIIB exhibits a strong substrate preference for the modified nucleotide 7-methylguanosine monophosphate but the structural reason for this preference was unknown. Here, we present crystal structures of cN-IIIB from Drosophila melanogaster bound to the reaction products 7-methylguanosine or cytidine. The structural data reveal that the cytosine- and 7-methylguanine moieties of the products are stacked between two aromatic residues in a coplanar but off-centered position. 7-methylguanosine is specifically bound through π-π interactions and distinguished from unmodified guanosine by additional cation-π coulomb interactions between the aromatic side chains and the positively charged 7-methylguanine. Notably, the base is further stabilized by T-shaped edge-to-face stacking of an additional tryptophan packing perpendicularly against the purine ring and forming, together with the other aromates, an aromatic slot. The structural data in combination with site-directed mutagenesis experiments reveal the molecular basis for the broad substrate specificity of cN-IIIB but also explain the substrate preference for 7-methylguanosine monophosphate. Analyzing the substrate specificities of cN-IIIB and the main pyrimidine 5′-nucleotidase cN-IIIA by mutagenesis studies, we show that cN-IIIA dephosphorylates the purine m7GMP as well, hence redefining its substrate spectrum. Docking calculations with cN-IIIA and m7GMP as well as biochemical data reveal that Asn69 does not generally exclude the turnover of purine substrates thus correcting previous suggestions. PMID:24603684

Comprehensive Evaluation for Substrate Selectivity of Cynomolgus Monkey Cytochrome P450 2C9, a New Efavirenz Oxidase.

PubMed

Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

2015-07-01

Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.

PubMed

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-07-29

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

PubMed Central

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-01-01

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. PMID:19680472

Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction.

PubMed

Wang, Hualei; Sun, Huihui; Wei, Dongzhi

2013-02-18

A nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(-)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(-)-mandelic acid without the unwanted byproduct is needed. A novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(-)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters Vmax, Km, Kcat, and Kcat/Km toward mandelonitrile were 45.4 μmol/min/mg, 0.14 mM, 15.4 s(-1), and 1.1×10(5) M(-1)s(-1), respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h. PESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(-)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(-)-mandelic acid in the industry.

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

PubMed

Hou, Jing; Zheng, Heping; Tzou, Wen-Shyong; Cooper, David R; Chruszcz, Maksymilian; Chordia, Mahendra D; Kwon, Keehwan; Grabowski, Marek; Minor, Wladek

2018-06-19

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of β-ketoacyl-ACP synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most Gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH and six vcFabH2 structures determined in either apo-form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters*

PubMed Central

Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky

2015-01-01

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393

Mesenchymal stem cell mechanobiology and emerging experimental platforms

PubMed Central

MacQueen, Luke; Sun, Yu; Simmons, Craig A.

2013-01-01

Experimental control over progenitor cell lineage specification can be achieved by modulating properties of the cell's microenvironment. These include physical properties of the cell adhesion substrate, such as rigidity, topography and deformation owing to dynamic mechanical forces. Multipotent mesenchymal stem cells (MSCs) generate contractile forces to sense and remodel their extracellular microenvironments and thereby obtain information that directs broad aspects of MSC function, including lineage specification. Various physical factors are important regulators of MSC function, but improved understanding of MSC mechanobiology requires novel experimental platforms. Engineers are bridging this gap by developing tools to control mechanical factors with improved precision and throughput, thereby enabling biological investigation of mechanics-driven MSC function. In this review, we introduce MSC mechanobiology and review emerging cell culture platforms that enable new insights into mechanobiological control of MSCs. Our main goals are to provide engineers and microtechnology developers with an up-to-date description of MSC mechanobiology that is relevant to the design of experimental platforms and to introduce biologists to these emerging platforms. PMID:23635493

Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

PubMed Central

Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

2014-01-01

Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

Substrate tunnels in enzymes: structure-function relationships and computational methodology.

PubMed

Kingsley, Laura J; Lill, Markus A

2015-04-01

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. © 2015 Wiley Periodicals, Inc.

Optimization of High-Throughput Sequencing Kinetics for determining enzymatic rate constants of thousands of RNA substrates

PubMed Central

Niland, Courtney N.; Jankowsky, Eckhard; Harris, Michael E.

2016-01-01

Quantification of the specificity of RNA binding proteins and RNA processing enzymes is essential to understanding their fundamental roles in biological processes. High Throughput Sequencing Kinetics (HTS-Kin) uses high throughput sequencing and internal competition kinetics to simultaneously monitor the processing rate constants of thousands of substrates by RNA processing enzymes. This technique has provided unprecedented insight into the substrate specificity of the tRNA processing endonuclease ribonuclease P. Here, we investigate the accuracy and robustness of measurements associated with each step of the HTS-Kin procedure. We examine the effect of substrate concentration on the observed rate constant, determine the optimal kinetic parameters, and provide guidelines for reducing error in amplification of the substrate population. Importantly, we find that high-throughput sequencing, and experimental reproducibility contribute their own sources of error, and these are the main sources of imprecision in the quantified results when otherwise optimized guidelines are followed. PMID:27296633

Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters

NASA Astrophysics Data System (ADS)

Zhao, Qin; Wang, Chengcheng; Wang, Chengyuan; Guo, Hui; Bao, Zhihao; Zhang, Minhua; Zhang, Peng

2015-07-01

Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

A comparative study of glycerol and sorbitol as co-substrates in methanol-induced cultures of Pichia pastoris: temperature effect and scale-up simulation.

PubMed

Berrios, Julio; Flores, María-Olga; Díaz-Barrera, Alvaro; Altamirano, Claudia; Martínez, Irene; Cabrera, Zaida

2017-03-01

The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either glycerol or sorbitol as co-substrate. Although both co-substrates have been widely used, comparative studies are scarce. In addition, these comparisons have been performed at different specific growth rate (µ) that it is well known that has an important effect on productivity. Thus, the effect of using these co-substrates on the production of Rhyzopus oryzae lipase (ROL) by P. pastoris was compared in continuous cultures growing at the same µ at either 22 or 30 °C. Results show that using glycerol as co-substrate led to higher volumetric productivities, and lower specific and volumetric methanol consumption rates. Scale-up simulation with 10-10,000 L bioreactor sizes indicated that glycerol produced the highest volumetric productivity of ROL with lower aeration requirements. Therefore, glycerol rises as a better option than sorbitol in ROL production.

PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

NASA Astrophysics Data System (ADS)

Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

2014-03-01

Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis.

PubMed

Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina; La Greca, Florencia; O'Donoghue, Anthony J; Schulze, Christopher J; Powers, James C; Craik, Charles S; Drag, Marcin; Jain, Sanjay K; Bogyo, Matthew

2016-11-11

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

PubMed Central

2016-01-01

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb “Hydrolase important for pathogenesis 1” (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections. PMID:27739665

Substrate Tunnels in Enzymes: Structure-Function Relationships and Computational Methodology

PubMed Central

Kingsley, Laura J.; Lill, Markus A.

2015-01-01

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein and in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years due to implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with tunnels and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. PMID:25663659

Mechanical Strength and Broadband Transparency Improvement of Glass Wafers via Surface Nanostructures.

PubMed

Kumar, Amarendra; Kashyap, Kunal; Hou, Max T; Yeh, J Andrew

2016-06-17

In this study, we mechanically strengthened a borosilicate glass wafer by doubling its bending strength and simultaneously enhancing its transparency using surface nanostructures for different applications including sensors, displays and panels. A fabrication method that combines dry and wet etching is used for surface nanostructure fabrication. Specifically, we improved the bending strength of plain borosilicate glass by 96% using these surface nanostructures on both sides. Besides bending strength improvement, a limited optical transmittance enhancement of 3% was also observed in the visible light wavelength region (400-800 nm). Both strength and transparency were improved by using surface nanostructures of 500 nm depth on both sides of the borosilicate glass without affecting its bulk properties or the glass manufacturing process. Moreover, we observed comparatively smaller fragments during the breaking of the nanostructured glass, which is indicative of strengthening. The range for the nanostructure depth is defined for different applications with which improvements of the strength and transparency of borosilicate glass substrate are obtained.

Mechanical Strength and Broadband Transparency Improvement of Glass Wafers via Surface Nanostructures

PubMed Central

Kumar, Amarendra; Kashyap, Kunal; Hou, Max T.; Yeh, J. Andrew

2016-01-01

In this study, we mechanically strengthened a borosilicate glass wafer by doubling its bending strength and simultaneously enhancing its transparency using surface nanostructures for different applications including sensors, displays and panels. A fabrication method that combines dry and wet etching is used for surface nanostructure fabrication. Specifically, we improved the bending strength of plain borosilicate glass by 96% using these surface nanostructures on both sides. Besides bending strength improvement, a limited optical transmittance enhancement of 3% was also observed in the visible light wavelength region (400–800 nm). Both strength and transparency were improved by using surface nanostructures of 500 nm depth on both sides of the borosilicate glass without affecting its bulk properties or the glass manufacturing process. Moreover, we observed comparatively smaller fragments during the breaking of the nanostructured glass, which is indicative of strengthening. The range for the nanostructure depth is defined for different applications with which improvements of the strength and transparency of borosilicate glass substrate are obtained. PMID:27322276

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

PubMed

Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

2015-07-24

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Knowledge-transfer learning for prediction of matrix metalloprotease substrate-cleavage sites.

PubMed

Wang, Yanan; Song, Jiangning; Marquez-Lago, Tatiana T; Leier, André; Li, Chen; Lithgow, Trevor; Webb, Geoffrey I; Shen, Hong-Bin

2017-07-18

Matrix Metalloproteases (MMPs) are an important family of proteases that play crucial roles in key cellular and disease processes. Therefore, MMPs constitute important targets for drug design, development and delivery. Advanced proteomic technologies have identified type-specific target substrates; however, the complete repertoire of MMP substrates remains uncharacterized. Indeed, computational prediction of substrate-cleavage sites associated with MMPs is a challenging problem. This holds especially true when considering MMPs with few experimentally verified cleavage sites, such as for MMP-2, -3, -7, and -8. To fill this gap, we propose a new knowledge-transfer computational framework which effectively utilizes the hidden shared knowledge from some MMP types to enhance predictions of other, distinct target substrate-cleavage sites. Our computational framework uses support vector machines combined with transfer machine learning and feature selection. To demonstrate the value of the model, we extracted a variety of substrate sequence-derived features and compared the performance of our method using both 5-fold cross-validation and independent tests. The results show that our transfer-learning-based method provides a robust performance, which is at least comparable to traditional feature-selection methods for prediction of MMP-2, -3, -7, -8, -9 and -12 substrate-cleavage sites on independent tests. The results also demonstrate that our proposed computational framework provides a useful alternative for the characterization of sequence-level determinants of MMP-substrate specificity.

Improved electrochemical properties of morphology-controlled titania/titanate nanostructures prepared by in-situ hydrothermal surface modification of self-source Ti substrate for high-performance supercapacitors.

PubMed

Banerjee, Arghya Narayan; Anitha, V C; Joo, Sang W

2017-10-16

Ti substrate surface is modified into two-dimensional (2D) TiO 2 nanoplatelet or one-dimensional (1D) nanorod/nanofiber (or a mixture of both) structure in a controlled manner via a simple KOH-based hydrothermal technique. Depending on the KOH concentration, different types of TiO 2 nanostructures (2D platelets, 1D nanorods/nanofibers and a 2D+1D mixed sample) are fabricated directly onto the Ti substrate surface. The novelty of this technique is the in-situ modification of the self-source Ti surface into titania nanostructures, and its direct use as the electrochemical microelectrode without any modifications. This leads to considerable improvement in the interfacial properties between metallic Ti and semiconducting TiO 2 . Since interfacial states/defects have profound effect on charge transport properties of electronic/electrochemical devices, therefore this near-defect-free interfacial property of Ti-TiO 2 microelectrode has shown high supercapacitive performances for superior charge-storage devices. Additionally, by hydrothermally tuning the morphology of titania nanostructures, the electrochemical properties of the electrodes are also tuned. A Ti-TiO 2 electrode comprising of a mixture of 2D-platelet+1D-nanorod structure reveals very high specific capacitance values (~7.4 mF.cm -2 ) due to the unique mixed morphology which manifests higher active sites (hence, higher utilization of the active materials) in terms of greater roughness at the 2D-platelet structures and higher surface-to-volume-ratio in the 1D-nanorod structures.

Macroinvertebrate community responses to gravel augmentation in a high-gradient, Southeastern regulated river

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

McManamay, Ryan A; Orth, Dr. Donald J; Dolloff, Dr. Charles A

2013-01-01

Sediment transport, one of the key processes of river systems, is altered or stopped by dams, leaving lower river reaches barren of sand and gravel, both of which are essential habitat for fish and macroinvertebrates. One way to compensate for losses in sediment is to supplement gravel to river reaches below impoundments. Because gravel addition has become a widespread practice, it is essential to evaluate the biotic response to restoration projects in order to improve the efficacy of future applications. The purpose of our study was to evaluate the response of the macroinvertebrate community to gravel addition in a high-gradient,more » regulated river in western North Carolina. We collected benthic macroinvertebrate samples from gravel-enhanced areas and unenhanced areas for 1 season before gravel addition, and for 4 seasons afterwards. Repeated measures multivariate analysis of variance indicated that the responses of macroinvertebrates to gravel addition were generally specific to individual taxa or particular functional feeding groups and did not lead to consistent patterns in overall family richness, diversity, density, or evenness. Non-metric multi-dimensional scaling showed that shifts in macroinvertebrate community composition were temporary and dependent upon site conditions and season. Correlations between macroinvertebrate response variables and substrate microhabitat variables existed with or without the inclusion of data from enhanced areas, which suggests that substrate-biotic relationships were present before gravel addition. A review of the current literature suggests that the responses of benthic macroinvertebrates to substrate restoration are inconsistent and dependent upon site conditions and the degree habitat improvement of pre-restoration site conditions.« less

Enhancement of the light output power of InGaN/GaN light-emitting diodes grown on pyramidal patterned sapphire substrates in the micro- and nanoscale

NASA Astrophysics Data System (ADS)

Gao, Haiyong; Yan, Fawang; Zhang, Yang; Li, Jinmin; Zeng, Yiping; Wang, Guohong

2008-01-01

Sapphire substrates were patterned by a chemical wet etching technique in the micro- and nanoscale to enhance the light output power of InGaN/GaN light-emitting diodes (LEDs). InGaN/GaN LEDs on a pyramidal patterned sapphire substrate in the microscale (MPSS) and pyramidal patterned sapphire substrate in the nanoscale (NPSS) were grown by metalorganic chemical vapor deposition. The characteristics of the LEDs fabricated on the MPSS and NPSS prepared by wet etching were studied and the light output powers of the LEDs fabricated on the MPSS and NPSS increased compared with that of the conventional LEDs fabricated on planar sapphire substrates. In comparison with the planar sapphire substrate, an enhancement in output power of about 29% and 48% is achieved with the MPSS and NPSS at an injection current of 20 mA, respectively. This significant enhancement is attributable to the improvement of the epitaxial quality of GaN-based epilayers and the improvement of the light extraction efficiency by patterned sapphire substrates. Additionally, the NPSS is more effective to enhance the light output power than the MPSS.

Resistive hydrogen sensing element

DOEpatents

Lauf, Robert J.

2000-01-01

Systems and methods are described for providing a hydrogen sensing element with a more robust exposed metallization by application of a discontinuous or porous overlay to hold the metallization firmly on the substrate. An apparatus includes: a substantially inert, electrically-insulating substrate; a first Pd containing metallization deposited upon the substrate and completely covered by a substantially hydrogen-impermeable layer so as to form a reference resistor on the substrate; a second Pd containing metallization deposited upon the substrate and at least a partially accessible to a gas to be tested, so as to form a hydrogen-sensing resistor; a protective structure disposed upon at least a portion of the second Pd containing metallization and at least a portion of the substrate to improve the attachment of the second Pd containing metallization to the substrate while allowing the gas to contact said the second Pd containing metallization; and a resistance bridge circuit coupled to both the first and second Pd containing metallizations. The circuit determines the difference in electrical resistance between the first and second Pd containing metallizations. The hydrogen concentration in the gas may be determined. The systems and methods provide advantages because adhesion is improved without adversely effecting measurement speed or sensitivity.

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

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

Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron

2013-04-01

The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes showmore » activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.« less

Exploring physical and chemical factors influencing the properties of recombinant prion protein and the real-time quaking-induced conversion (RT-QuIC) assay.

PubMed

Cheng, Keding; Sloan, Angela; Avery, Kristen M; Coulthart, Michael; Carpenter, Michael; Knox, J David

2014-01-01

Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP(d)) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.

Hydrogen plasma treatment for improved conductivity in amorphous aluminum doped zinc tin oxide thin films

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

Morales-Masis, M., E-mail: monica.moralesmasis@epfl.ch; Ding, L.; Dauzou, F.

2014-09-01

Improving the conductivity of earth-abundant transparent conductive oxides (TCOs) remains an important challenge that will facilitate the replacement of indium-based TCOs. Here, we show that a hydrogen (H{sub 2})-plasma post-deposition treatment improves the conductivity of amorphous aluminum-doped zinc tin oxide while retaining its low optical absorption. We found that the H{sub 2}-plasma treatment performed at a substrate temperature of 50 °C reduces the resistivity of the films by 57% and increases the absorptance by only 2%. Additionally, the low substrate temperature delays the known formation of tin particles with the plasma and it allows the application of the process to temperature-sensitivemore » substrates.« less

Improved output power of GaN-based light-emitting diodes grown on a nanopatterned sapphire substrate

NASA Astrophysics Data System (ADS)

Chan, Chia-Hua; Hou, Chia-Hung; Tseng, Shao-Ze; Chen, Tsing-Jen; Chien, Hung-Ta; Hsiao, Fu-Li; Lee, Chien-Chieh; Tsai, Yen-Ling; Chen, Chii-Chang

2009-07-01

This letter describes the improved output power of GaN-based light-emitting diodes (LEDs) formed on a nanopatterned sapphire substrate (NPSS) prepared through etching with a self-assembled monolayer of 750-nm-diameter SiO2 nanospheres used as the mask. The output power of NPSS LEDs was 76% greater than that of LEDs on a flat sapphire substrate. Three-dimensional finite-difference time-domain calculation predicted a 40% enhancement in light extraction efficiency of NPSS LEDs. In addition, the reduction of full widths at half maximum in the ω-scan rocking curves for the (0 0 2) and (1 0 2) planes of GaN on NPSS suggested improved crystal quality.

Tribology of nitrided-coated steel-a review

NASA Astrophysics Data System (ADS)

Bhaskar, Santosh V.; Kudal, Hari N.

2017-01-01

Surface engineering such as surface treatment, coating, and surface modification are employed to increase surface hardness, minimize adhesion, and hence, to reduce friction and improve resistance to wear. To have optimal tribological performance of Physical Vapor Deposition (PVD) hard coating to the substrate materials, pretreatment of the substrate materials is always advisable to avoid plastic deformation of the substrate, which may result in eventual coating failure. The surface treatment results in hardening of the substrate and increase in load support effect. Many approaches aim to improve the adhesion of the coatings onto the substrate and nitriding is the one of the best suitable options for the same. In addition to tribological properties, nitriding leads to improved corrosion resistance. Often corrosion resistance is better than that obtainable with other surface engineering processes such as hard-chrome and nickel plating. Ability of this layer to withstand thermal stresses gives stability which extends the surface life of tools and other components exposed to heat. Most importantly, the nitrogen picked-up by the diffusion layer increases the rotating-bending fatigue strength in components. The present article reviews mainly the tribological advancement of different nitrided-coated steels based on the types of coatings, structure, and the tribo-testing parameters, in recent years.

Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

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

Critton, D.; Tortajada, A; Stetson, G

2008-01-01

Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

PubMed Central

Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

2016-01-01

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages. PMID:27768715

Oxidase-functionalized Fe(3)O(4) nanoparticles for fluorescence sensing of specific substrate.

PubMed

Liu, Cheng-Hao; Tseng, Wei-Lung

2011-10-03

This study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe(3)O(4) nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H(2)O(2), the reaction pH between Fe(3)O(4) NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe(3)O(4) NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe(3)O(4) NPs and oxidase for the quantification of specific substrates through the H(2)O(2)-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 μM using glucose oxidase-Fe(3)O(4), galactose oxidase-Fe(3)O(4), and choline oxidase-Fe(3)O(4) composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. Copyright © 2011 Elsevier B.V. All rights reserved.

Isozyme-specific comprehensive characterization of transglutaminase-crosslinked substrates in kidney fibrosis.

PubMed

Tatsukawa, Hideki; Otsu, Risa; Tani, Yuji; Wakita, Ryosuke; Hitomi, Kiyotaka

2018-05-09

Chronic kidney disease is characterized by prolonged decline in renal function, excessive accumulation of ECM, and progressive tissue fibrosis. Transglutaminase (TG) is a crosslinking enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues, and is involved in the induction of renal fibrosis via the stabilization of ECM and the activation of TGF-β1. Despite the accumulating evidences indicating that TG2 is a key enzyme in fibrosis, genetic knockout of TG2 reduced by only 50% the elevated protein crosslinking and fibrous protein in renal fibrosis model, whereas treatment with TG inhibitor almost completely reduced these levels. Here, we also clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific crosslinked substrates for both TG1 and TG2 in fibrotic kidney. We found that TG1 activity was markedly enhanced in renal tubular epithelium and interstitial areas, whereas TG2 activity increased only in the extracellular space. In total, 47 and 67 possible candidates were identified as TG1 and TG2 substrates, respectively, only in fibrotic kidney. Among them, several possible substrates related to renal disease and fibrosis were identified. These findings provide novel insights into the mechanisms of renal fibrosis through the targeting of isozyme-specific TG substrates.