The role of an active site Mg(2+) in HDV ribozyme self-cleavage: insights from QM/MM calculations.

PubMed

Mlýnský, Vojtěch; Walter, Nils G; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

2015-01-07

The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H(+) in the active site, which acts as the general acid, and a partially hydrated Mg(2+) ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with a distinct position and coordination of the catalytically important active site Mg(2+) ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal mol(-1), indicating that the specific position of the Mg(2+) ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2'-OH) nucleophile and the nucleophilic attack of the resulting U-1(2'-O(-)) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimate the pKa of the U-1(2'-OH) group to range from 8.8 to 11.2, suggesting that it is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg(2+) ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg(2+) ion, facilitate deprotonation and activation of the 2'-OH nucleophile.

Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

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

Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.

2015-06-04

Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiO x domains are titrated on TiO x–SiO 2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiO x, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H 2O 2 activation occurs. We use this method to determine the active sitemore » densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h -1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.« less

Spectroscopic insights into the nature of active sites in iron–nitrogen–carbon electrocatalysts for oxygen reduction in acid

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

Jia, Qingying; Ramaswamy, Nagappan; Tylus, Urszula

Developing efficient and inexpensive catalysts for the sluggish oxygen reduction reaction (ORR) constitutes one of the grand challenges in the fabrication of commercially viable fuel cell devices and metal–air batteries for future energy applications. Despite recent achievements in designing advanced Pt-based and Pt-free catalysts, current progress primarily involves an empirical approach of trial-and-error combination of precursors and synthesis conditions, which limits further progress. Rational design of catalyst materials requires proper understanding of the mechanistic origin of the ORR and the underlying surface properties under operating conditions that govern catalytic activity. Herein, several different groups of iron-based catalysts synthesized via differentmore » methods and/or precursors were systematically studied by combining multiple spectroscopic techniques under ex situ and in situ conditions in an effort to obtain a comprehensive understanding of the synthesis-products correlations, nature of active sites, and the reaction mechanisms. These catalysts include original macrocycles, macrocycle-pyrolyzed catalysts, and Fe-N–C catalysts synthesized from individual Fe, N, and C precursors including polymer-based catalysts, metal organic framework (MOF)-based catalysts, and sacrificial support method (SSM)-based catalysts. The latter group of catalysts is most promising as not only they exhibit exceptional ORR activity and/or durability, but also the final products are controllable. We show that the high activity observed for most pyrolyzed Fe-based catalysts can mainly be attributed to a single active site: non-planar Fe–N 4 moiety embedded in distorted carbon matrix characterized by a high potential for the Fe 2+/3+ redox transition in acidic electrolyte/environment. The high intrinsic ORR activity, or turnover frequency (TOF), of this site is shown to be accounted for by redox catalysis mechanism that highlights the dominant role of the site

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

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

O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

DOE PAGES

O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

2016-12-22

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

CASPASE-9 CARD:CORE DOMAIN INTERACTIONS REQUIRE A PROPERLY-FORMED ACTIVE SITE

PubMed Central

Huber, Kristen L.; Serrano, Banyuhay P.; Hardy, Jeanne A.

2018-01-01

Caspase-9 is a critical factor in the initiation of apoptosis, and as a result is tightly regulated by a number of mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Together these findings provide mechanistic insight into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate. PMID:29500231

Insights into GATA-1 Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

PubMed Central

Yu, Ming; Riva, Laura; Xie, Huafeng; Schindler, Yocheved; Moran, Tyler B.; Cheng, Yong; Yu, Duonan; Hardison, Ross; Weiss, Mitchell J; Orkin, Stuart H.; Bernstein, Bradley E.; Fraenkel, Ernest; Cantor, Alan B.

2009-01-01

Summary The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo. PMID:19941827

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

PubMed Central

Lucas, James E; Siegel, Justin B

2015-01-01

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

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

Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putativemore » nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.« less

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

PubMed

Ali, Abid; Azam, Mohd W; Khan, Asad U

2018-06-01

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE PAGES

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

2017-07-07

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Direct atomic-level insight into the active sites of a high-performance PGM-free ORR catalyst

NASA Astrophysics Data System (ADS)

Chung, Hoon T.; Cullen, David A.; Higgins, Drew; Sneed, Brian T.; Holby, Edward F.; More, Karren L.; Zelenay, Piotr

2017-08-01

Platinum group metal-free (PGM-free) metal-nitrogen-carbon catalysts have emerged as a promising alternative to their costly platinum (Pt)-based counterparts in polymer electrolyte fuel cells (PEFCs) but still face some major challenges, including (i) the identification of the most relevant catalytic site for the oxygen reduction reaction (ORR) and (ii) demonstration of competitive PEFC performance under automotive-application conditions in the hydrogen (H2)-air fuel cell. Herein, we demonstrate H2-air performance gains achieved with an iron-nitrogen-carbon catalyst synthesized with two nitrogen precursors that developed hierarchical porosity. Current densities recorded in the kinetic region of cathode operation, at fuel cell voltages greater than ~0.75 V, were the same as those obtained with a Pt cathode at a loading of 0.1 milligram of Pt per centimeter squared. The proposed catalytic active site, carbon-embedded nitrogen-coordinated iron (FeN4), was directly visualized with aberration-corrected scanning transmission electron microscopy, and the contributions of these active sites associated with specific lattice-level carbon structures were explored computationally.

Direct atomic-level insight into the active sites of a high-performance PGM-free ORR catalyst

DOE PAGES

Chung, Hoon T.; Cullen, David A.; Higgins, Drew; ...

2017-08-04

Platinum group metal–free (PGM-free) metal-nitrogen-carbon catalysts have emerged as a promising alternative to their costly platinum (Pt)–based counterparts in polymer electrolyte fuel cells (PEFCs) but still face some major challenges, including (i) the identification of the most relevant catalytic site for the oxygen reduction reaction (ORR) and (ii) demonstration of competitive PEFC performance under automotive-application conditions in the hydrogen (H 2)–air fuel cell. We demonstrate H 2-air performance gains achieved with an iron-nitrogen-carbon catalyst synthesized with two nitrogen precursors that developed hierarchical porosity. In current densities recorded in the kinetic region of cathode operation, at fuel cell voltages greater thanmore » ~0.75 V, were the same as those obtained with a Pt cathode at a loading of 0.1 milligram of Pt per centimeter squared. The catalytic active site we proposed, carbon-embedded nitrogen-coordinated iron (FeN 4), was directly visualized with aberration-corrected scanning transmission electron microscopy, and the contributions of these active sites associated with specific lattice-level carbon structures were explored computationally.« less

Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form.

PubMed

Lee, Yie-Vern; Choi, Sy Bing; Wahab, Habibah A; Choong, Yee Siew

2017-09-25

Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C 2 -C 3 bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.

Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

PubMed

Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

2016-08-01

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Directing reaction pathways by catalyst active-site selection using self-assembled monolayers.

PubMed

Pang, Simon H; Schoenbaum, Carolyn A; Schwartz, Daniel K; Medlin, J Will

2013-01-01

One key route for controlling reaction selectivity in heterogeneous catalysis is to prepare catalysts that exhibit only specific types of sites required for desired product formation. Here we show that alkanethiolate self-assembled monolayers with varying surface densities can be used to tune selectivity to desired hydrogenation and hydrodeoxygenation products during the reaction of furfural on supported palladium catalysts. Vibrational spectroscopic studies demonstrate that the selectivity improvement is achieved by controlling the availability of specific sites for the hydrogenation of furfural on supported palladium catalysts through the selection of an appropriate alkanethiolate. Increasing self-assembled monolayer density by controlling the steric bulk of the organic tail ligand restricts adsorption on terrace sites and dramatically increases selectivity to desired products furfuryl alcohol and methylfuran. This technique of active-site selection simultaneously serves both to enhance selectivity and provide insight into the reaction mechanism.

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

PubMed

Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

2016-06-28

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

PubMed

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-05-01

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.

Selection of the InSight landing site

USGS Publications Warehouse

Golombek, M.; Kipp, D.; Warner, N.; Daubar, Ingrid J.; Fergason, Robin L.; Kirk, Randolph L.; Beyer, R.; Huertas, A.; Piqueux, Sylvain; Putzig, N.E.; Campbell, B.A.; Morgan, G. A.; Charalambous, C.; Pike, W. T.; Gwinner, K.; Calef, F.; Kass, D.; Mischna, M A; Ashley, J.; Bloom, C.; Wigton, N.; Hare, T.; Schwartz, C.; Gengl, H.; Redmond, L.; Trautman, M.; Sweeney, J.; Grima, C.; Smith, I. B.; Sklyanskiy, E.; Lisano, M.; Benardini, J.; Smrekar, S.E.; Lognonne, P.; Banerdt, W. B.

2017-01-01

The selection of the Discovery Program InSight landing site took over four years from initial identification of possible areas that met engineering constraints, to downselection via targeted data from orbiters (especially Mars Reconnaissance Orbiter (MRO) Context Camera (CTX) and High-Resolution Imaging Science Experiment (HiRISE) images), to selection and certification via sophisticated entry, descent and landing (EDL) simulations. Constraints on elevation (≤−2.5 km">≤−2.5 km≤−2.5 km for sufficient atmosphere to slow the lander), latitude (initially 15°S–5°N and later 3°N–5°N for solar power and thermal management of the spacecraft), ellipse size (130 km by 27 km from ballistic entry and descent), and a load bearing surface without thick deposits of dust, severely limited acceptable areas to western Elysium Planitia. Within this area, 16 prospective ellipses were identified, which lie ∼600 km north of the Mars Science Laboratory (MSL) rover. Mapping of terrains in rapidly acquired CTX images identified especially benign smooth terrain and led to the downselection to four northern ellipses. Acquisition of nearly continuous HiRISE, additional Thermal Emission Imaging System (THEMIS), and High Resolution Stereo Camera (HRSC) images, along with radar data confirmed that ellipse E9 met all landing site constraints: with slopes <15° at 84 m and 2 m length scales for radar tracking and touchdown stability, low rock abundance (<10 %) to avoid impact and spacecraft tip over, instrument deployment constraints, which included identical slope and rock abundance constraints, a radar reflective and load bearing surface, and a fragmented regolith ∼5 m thick for full penetration of the heat flow probe. Unlike other Mars landers, science objectives did not directly influence landing site selection.

Real-Time Visualization of Active Species in a Single-Site Metal–Organic Framework Photocatalyst

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

Yang, Sizhuo; Pattengale, Brian; Lee, Sungsik

In this work, we report a new single-site photocatalyst (Co-Ru-UIO- 67(bpy)) based on a metal-organic framework platform with incorporated molecular photosensitizer and catalyst. We show that this catalyst not only demonstrates exceptional activity for light-driven H2 production but also can be recycled without loss of activity. Using the combination of optical transient absorption spectroscopy and in situ X-ray absorption spectroscopy, we not only captured the key CoI intermediate species formed after ultrafast charge transfer from the incorporated photosensitizer but also identified the rate-limiting step in the catalytic cycle, providing insight into the catalysis mechanism of these single-site metal-organic framework photocatalysts.

XET Activity is Found Near Sites of Growth and Cell Elongation in Bryophytes and Some Green Algae: New Insights into the Evolution of Primary Cell Wall Elongation

PubMed Central

Van Sandt, Vicky S. T.; Stieperaere, Herman; Guisez, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

2007-01-01

Background and Aims In angiosperms xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is involved in reorganization of the cell wall during growth and development. The location of oligo-xyloglucan transglucosylation activity and the presence of XTH expressed sequence tags (ESTs) in the earliest diverging extant plants, i.e. in bryophytes and algae, down to the Phaeophyta was examined. The results provide information on the presence of an XET growth mechanism in bryophytes and algae and contribute to the understanding of the evolution of cell wall elongation in general. Methods Representatives of the different plant lineages were pressed onto an XET test paper and assayed. XET or XET-related activity was visualized as the incorporation of fluorescent signal. The Physcomitrella genome database was screened for the presence of XTHs. In addition, using the 3′ RACE technique searches were made for the presence of possible XTH ESTs in the Charophyta. Key Results XET activity was found in the three major divisions of bryophytes at sites corresponding to growing regions. In the Physcomitrella genome two putative XTH-encoding cDNA sequences were identified that contain all domains crucial for XET activity. Furthermore, XET activity was located at the sites of growth in Chara (Charophyta) and Ulva (Chlorophyta) and a putative XTH ancestral enzyme in Chara was identified. No XET activity was identified in the Rhodophyta or Phaeophyta. Conclusions XET activity was shown to be present in all major groups of green plants. These data suggest that an XET-related growth mechanism originated before the evolutionary divergence of the Chlorobionta and open new insights in the evolution of the mechanisms of primary cell wall expansion. PMID:17098750

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

PubMed

Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

2016-04-01

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation.

PubMed

Cobbaut, Mathias; Derua, Rita; Parker, Peter J; Waelkens, Etienne; Janssens, Veerle; Van Lint, Johan

2018-06-22

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P+1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

PubMed

Skov, Lars K; Pizzut-Serin, Sandra; Remaud-Simeon, Magali; Ernst, Heidi A; Gajhede, Michael; Mirza, Osman

2013-09-01

Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

PubMed Central

Hytönen, Vesa P; Vogel, Viola

2008-01-01

Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

Parkin-phosphoubiquitin complex reveals a cryptic ubiquitin binding site required for RBR ligase activity

PubMed Central

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-01-01

RING-BETWEENRING-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR is Parkin, mutations in which lead to early onset hereditary Parkinsonism. Parkin and other RBRs share a catalytic RBR module, but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during activation of Parkin. However, current data on active RBRs are in the absence of regulatory domains. Therefore, how individual RBRs are activated, and whether they share a common mechanism remains unclear. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces a movement in the IBR domain to reveal a cryptic ubiquitin binding site. Mutation of this site negatively impacts on Parkin’s activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, suggesting a role for interdomain association in RBR ligase mechanism. PMID:28414322

A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria.

PubMed

Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng; Gu, Yang; Jiang, Weihong

2017-01-24

Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named cre var , has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of cre var can affect CcpA binding affinity, and moreover, the core palindromic sequence of cre var is the key structure for regulation. Such a variable architecture of cre var shows potential importance for CcpA's diverse and fine regulation. A total of 103 potential cre var sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 cre var sites are associated with 27 genes involved in many important pathways. Also of significance, the cre var sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria. In Gram-positive bacteria, the global regulator CcpA controls a large number of important physiological and metabolic processes. Although a typical consensus CcpA-binding site, cre, has been identified, it remains

Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

PubMed

Adamczyk, Katrin; Simpson, Niall; Greetham, Gregory M; Gumiero, Andrea; Walsh, Martin A; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

2015-01-01

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H 2 O and D 2 O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump-probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.

Roles of s3 site residues of nattokinase on its activity and substrate specificity.

PubMed

Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

2007-09-01

Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

Analysis of Local Slopes at the InSight Landing Site on Mars

NASA Astrophysics Data System (ADS)

Fergason, R. L.; Kirk, R. L.; Cushing, G.; Galuszka, D. M.; Golombek, M. P.; Hare, T. M.; Howington-Kraus, E.; Kipp, D. M.; Redding, B. L.

2017-10-01

To evaluate the topography of the surface within the InSight candidate landing ellipses, we generated Digital Terrain Models (DTMs) at lander scales and those appropriate for entry, descent, and landing simulations, along with orthoimages of both images in each stereopair, and adirectional slope images. These products were used to assess the distribution of slopes for each candidate ellipse and terrain type in the landing site region, paying particular attention to how these slopes impact InSight landing and engineering safety, and results are reported here. Overall, this region has extremely low slopes at 1-meter baseline scales and meets the safety constraints of the InSight lander. The majority of the landing ellipse has a mean slope at 1-meter baselines of 3.2°. In addition, a mosaic of HRSC, CTX, and HiRISE DTMs within the final landing ellipse (ellipse 9) was generated to support entry, descent, and landing simulations and evaluations. Several methods were tested to generate this mosaic and the NASA Ames Stereo Pipeline program dem_mosaic produced the best results. For the HRSC-CTX-HiRISE DTM mosaic, more than 99 % of the mosaic has slopes less than 15°, and the introduction of artificially high slopes along image seams was minimized.

Analysis of local slopes at the InSight landing site on Mars

USGS Publications Warehouse

Fergason, Robin L.; Kirk, Randolph L.; Cushing, Glen; Galuszka, Donna M.; Golombek, Matthew P.; Hare, Trent M.; Howington-Kraus, Elpitha; Kipp, Devin M; Redding, Bonnie L.

2017-01-01

To evaluate the topography of the surface within the InSight candidate landing ellipses, we generated Digital Terrain Models (DTMs) at lander scales and those appropriate for entry, descent, and landing simulations, along with orthoimages of both images in each stereopair, and adirectional slope images. These products were used to assess the distribution of slopes for each candidate ellipse and terrain type in the landing site region, paying particular attention to how these slopes impact InSight landing and engineering safety, and results are reported here. Overall, this region has extremely low slopes at 1-meter baseline scales and meets the safety constraints of the InSight lander. The majority of the landing ellipse has a mean slope at 1-meter baselines of 3.2°. In addition, a mosaic of HRSC, CTX, and HiRISE DTMs within the final landing ellipse (ellipse 9) was generated to support entry, descent, and landing simulations and evaluations. Several methods were tested to generate this mosaic and the NASA Ames Stereo Pipeline program dem_mosaic produced the best results. For the HRSC-CTX-HiRISE DTM mosaic, more than 99 % of the mosaic has slopes less than 15°, and the introduction of artificially high slopes along image seams was minimized.

Normal Modes Expose Active Sites in Enzymes.

PubMed

Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

2016-12-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.

Normal Modes Expose Active Sites in Enzymes

PubMed Central

Glantz-Gashai, Yitav; Samson, Abraham O.

2016-01-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

PubMed

Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

2018-05-30

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Ni-Nanocluster Modified Black TiO2 with Dual Active Sites for Selective Photocatalytic CO2 Reduction.

PubMed

Billo, Tadesse; Fu, Fang-Yu; Raghunath, Putikam; Shown, Indrajit; Chen, Wei-Fu; Lien, Hsiang-Ting; Shen, Tzu-Hsien; Lee, Jyh-Fu; Chan, Ting-Shan; Huang, Kuo-You; Wu, Chih-I; Lin, M C; Hwang, Jih-Shang; Lee, Chih-Hao; Chen, Li-Chyong; Chen, Kuei-Hsien

2018-01-01

One of the key challenges in artificial photosynthesis is to design a photocatalyst that can bind and activate the CO 2 molecule with the smallest possible activation energy and produce selective hydrocarbon products. In this contribution, a combined experimental and computational study on Ni-nanocluster loaded black TiO 2 (Ni/TiO 2[Vo] ) with built-in dual active sites for selective photocatalytic CO 2 conversion is reported. The findings reveal that the synergistic effects of deliberately induced Ni nanoclusters and oxygen vacancies provide (1) energetically stable CO 2 binding sites with the lowest activation energy (0.08 eV), (2) highly reactive sites, (3) a fast electron transfer pathway, and (4) enhanced light harvesting by lowering the bandgap. The Ni/TiO 2[Vo] photocatalyst has demonstrated highly selective and enhanced photocatalytic activity of more than 18 times higher solar fuel production than the commercial TiO 2 (P-25). An insight into the mechanisms of interfacial charge transfer and product formation is explored. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

PubMed

Pedroso, Marcelo M; Ely, Fernanda; Carpenter, Margaret C; Mitić, Nataša; Gahan, Lawrence R; Ollis, David L; Wilcox, Dean E; Schenk, Gerhard

2017-07-05

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its β site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the β site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the β site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the β site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

PubMed

Jiang, Xukai; Wang, Yuying; Xu, Limei; Chen, Guanjun; Wang, Lushan

2017-09-09

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

PubMed Central

Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

2015-01-01

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

DOE PAGES

Sunden, Fanny; Peck, Ariana; Salzman, Julia; ...

2015-04-22

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

PubMed

Bihani, Subhash C; Chakravarty, Dhiman; Ballal, Anand

2016-04-01

Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes.

PubMed

Suplatov, D A; Arzhanik, V K; Svedas, V K

2011-01-01

Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the

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

PubMed Central

Dreveny, Ingrid; Kratky, Christoph; Gruber, Karl

2002-01-01

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

Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

PubMed

Lenz, Stefan A P; Wetmore, Stacey D

2016-02-09

different DNA-protein interactions used by AAG to facilitate BER, as well as the crucial role of the general base and water (nucleophile) positioning. The insights gained from our work will aid the understanding of the function of other enzymes that use flexible active sites to exhibit diverse substrate specificity.

Dissecting the active site of a photoreceptor protein

NASA Astrophysics Data System (ADS)

Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

Influences of Media Compositions on Characteristics of Isolated Bacteria Exhibiting Lignocellulolytic Activities from Various Environmental Sites.

PubMed

Gong, Gyeongtaek; Lee, Sun-Mi; Woo, Han Min; Park, Tai Hyun; Um, Youngsoon

2017-11-01

Efficient isolation of lignocellulolytic bacteria is essential for the utilization of lignocellulosic biomass. In this study, bacteria with cellulolytic, xylanolytic, and lignolytic activities were isolated from environmental sites such as mountain, wetland, and mudflat using isolation media containing the combination of lignocellulose components (cellulose, xylan, and lignin). Eighty-nine isolates from the isolation media were characterized by analyzing taxonomic ranks and cellulolytic, xylanolytic, and lignolytic activities. Most of the cellulolytic bacteria showed multienzymatic activities including xylanolytic activity. The isolation media without lignin were efficient in isolating bacteria exhibiting multienzymatic activities even including lignolytic activity, whereas a lignin-containing medium was effective to isolate bacteria exhibiting lignolytic activity only. Multienzymatic activities were mainly observed in Bacillus and Streptomyces, while Burkholderia was the most abundant genus with lignolytic activity only. This study provides insight into isolation medium for efficient isolation of lignocellulose-degrading microorganisms.

A contact photo-cross-linking investigation of the active site of the 8-17 deoxyribozyme.

PubMed

Liu, Yong; Sen, Dipankar

2008-09-12

The small RNA-cleaving 8-17 deoxyribozyme (DNAzyme) has been the subject of extensive mechanistic and structural investigation, including a number of recent single-molecule studies of its global folding. Little detailed insight exists, however, into this DNAzyme's active site; for instance, the identity of specific nucleotides that are proximal to or in contact with the scissile site in the substrate. Here, we report a systematic replacement of a number of bases within the magnesium-folded DNAzyme-substrate complex with thio- and halogen-substituted base analogues, which were then photochemically activated to generate contact cross-links within the complex. Mapping of the cross-links revealed a striking pattern of DNAzyme-substrate cross-links but an absence of significant intra-DNAzyme cross-links. Notably, the two nucleotides directly flanking the scissile phosphodiester cross-linked strongly with functionally important elements within the DNAzyme, the thymine of a G.T wobble base pair, a WCGR bulge loop, and a terminal AGC loop. Mutation of the wobble base pair to a G-C pair led to a significant folding instability of the DNAzyme-substrate complex. The cross-linking patterns obtained were used to generate a model for the DNAzyme's active site that had the substrate's scissile phosphodiester sandwiched between the DNAzyme's wobble thymine and its AGC and WCGR loops.

Active site dynamics of ribonuclease.

PubMed Central

Brünger, A T; Brooks, C L; Karplus, M

1985-01-01

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state. Images PMID:3866234

Radar interferometry offers new insights into threats to the Angkor site.

PubMed

Chen, Fulong; Guo, Huadong; Ma, Peifeng; Lin, Hui; Wang, Cheng; Ishwaran, Natarajan; Hang, Peou

2017-03-01

The conservation of World Heritage is critical to the cultural and social sustainability of regions and nations. Risk monitoring and preventive diagnosis of threats to heritage sites in any given ecosystem are a complex and challenging task. Taking advantage of the performance of Earth Observation technologies, we measured the impacts of hitherto imperceptible and poorly understood factors of groundwater and temperature variations on the monuments in the Angkor World Heritage site (400 km 2 ). We developed a two-scale synthetic aperture radar interferometry (InSAR) approach. We describe spatial-temporal displacements (at millimeter-level accuracy), as measured by high-resolution TerraSAR/TanDEM-X satellite images, to provide a new solution to resolve the current controversy surrounding the potential structural collapse of monuments in Angkor. Multidisciplinary analysis in conjunction with a deterioration kinetics model offers new insights into the causes that trigger the potential decline of Angkor monuments. Our results show that pumping groundwater for residential and touristic establishments did not threaten the sustainability of monuments during 2011 to 2013; however, seasonal variations of the groundwater table and the thermodynamics of stone materials are factors that could trigger and/or aggravate the deterioration of monuments. These factors amplify known impacts of chemical weathering and biological alteration of temple materials. The InSAR solution reported in this study could have implications for monitoring and sustainable conservation of monuments in World Heritage sites elsewhere.

Radar interferometry offers new insights into threats to the Angkor site

PubMed Central

Chen, Fulong; Guo, Huadong; Ma, Peifeng; Lin, Hui; Wang, Cheng; Ishwaran, Natarajan; Hang, Peou

2017-01-01

The conservation of World Heritage is critical to the cultural and social sustainability of regions and nations. Risk monitoring and preventive diagnosis of threats to heritage sites in any given ecosystem are a complex and challenging task. Taking advantage of the performance of Earth Observation technologies, we measured the impacts of hitherto imperceptible and poorly understood factors of groundwater and temperature variations on the monuments in the Angkor World Heritage site (400 km2). We developed a two-scale synthetic aperture radar interferometry (InSAR) approach. We describe spatial-temporal displacements (at millimeter-level accuracy), as measured by high-resolution TerraSAR/TanDEM-X satellite images, to provide a new solution to resolve the current controversy surrounding the potential structural collapse of monuments in Angkor. Multidisciplinary analysis in conjunction with a deterioration kinetics model offers new insights into the causes that trigger the potential decline of Angkor monuments. Our results show that pumping groundwater for residential and touristic establishments did not threaten the sustainability of monuments during 2011 to 2013; however, seasonal variations of the groundwater table and the thermodynamics of stone materials are factors that could trigger and/or aggravate the deterioration of monuments. These factors amplify known impacts of chemical weathering and biological alteration of temple materials. The InSAR solution reported in this study could have implications for monitoring and sustainable conservation of monuments in World Heritage sites elsewhere. PMID:28275729

Low dielectric response in enzyme active site

PubMed Central

Mertz, Edward L.; Krishtalik, Lev I.

2000-01-01

The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

Low Mitochondrial DNA Diversity in an Ancient Population from China: Insight into Social Organization at the Fujia Site.

PubMed

Dong, Yu; Li, Chunxiang; Luan, Fengshi; Li, Zhenguang; Li, Hongjie; Cui, Yinqiu; Zhou, Hui; Malhi, Ripan S

2015-01-01

To gain insight into the social organization of a population associated with the Dawenkou period, we performed ancient DNA analysis of 18 individuals from human remains from the Fujia site in Shandong Province, China. Directly radiocarbon dated to 4800-4500 cal BP, the Fujia site is assumed to be associated with a transitional phase from matrilineal clans to patrilineal monogamous families. Our results reveal a low mitochondrial DNA diversity from the site and population. Combined with Y chromosome data, the pattern observed at the Fujia site is most consistent with a matrilineal community. The patterns also suggest that the bond of marriage was de-emphasized compared with the bonds of descent at Fujia.

Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

PubMed Central

Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

2016-01-01

Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

PubMed

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-05-03

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

Comparative structural modeling of six old yellow enzymes (OYEs) from the necrotrophic fungus Ascochyta rabiei: insight into novel OYE classes with differences in cofactor binding, organization of active site residues and stereopreferences.

PubMed

Nizam, Shadab; Gazara, Rajesh Kumar; Verma, Sandhya; Singh, Kunal; Verma, Praveen Kumar

2014-01-01

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.

Dynamics of active sites in biological macromolecules using a Green-function approach: An application to heme vibrational dynamics in myoglobin

NASA Astrophysics Data System (ADS)

Rai, Brajesh; Prohofsky, Earl

2003-03-01

Dynamics of functionally active regions of biological macromolecules can be studied using a Green-function technique. This approach uses the fact that in most cases one has a good set of force constants for active sites, and rather poorly defined force field parameters for other regions of the macromolecule. The Green-function method is applied to study the iron vibrational modes of the heme active site in myoglobin. In this approach, the heme active site is viewed as a system interacting with surrounding globin, which acts as an excitation bath. The normal modes of heme and globin are separately calculated using the best available force fields for the two entities. The iron vibrational spectrum of myoglobin is then obtained using the solutions of the heme and globin, and by considering physically meaningful interactions between the two units. The refinement of the Green-function calculations to the experimental data from an x-ray synchrotron-based Nuclear Resonance Vibrational Spectroscopy provides important insights into the character of iron normal modes of myoglobin.

Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

DOE PAGES

Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

2014-12-09

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the

Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes.

PubMed

Geeraerts, Zachary; Celis, Arianna I; Mayfield, Jeffery A; Lorenz, Megan; Rodgers, Kenton R; DuBois, Jennifer L; Lukat-Rodgers, Gudrun S

2018-03-06

O 2 -evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO 2 - ) to O 2 and Cl - . Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO 2 - , albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10 -9 M, the K D for the KpCld-CN - complex is 2 orders of magnitude smaller than that of DaCld-CN - and indicates an affinity for CN - that is greater than that of most heme proteins. The difference in CN - affinity between Kp- and DaClds is predominantly due to differences in k off . The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p K a of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 396 cm -1 ) and KpCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 356 cm -1 ) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

PubMed

Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

2014-12-16

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

PubMed Central

2015-01-01

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

Non-competitive inhibition by active site binders.

PubMed

Blat, Yuval

2010-06-01

Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.

Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

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

Zull, Lawrence M.; Yeniscavich, William

2008-01-15

The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive materialmore » contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.« less

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

PubMed

Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

2017-07-21

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

PubMed Central

Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

2013-01-01

Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.

PubMed

Rodrigues, Andria V; Kandegedara, Ashoka; Rotondo, John A; Dancis, Andrew; Stemmler, Timothy L

2015-06-01

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

A pharmacophore model specific to active site of CYP1A2 with a novel molecular modeling explorer and CoMFA.

PubMed

Zhang, Tao; Wei, Dong-Qing; Chou, Kuo-Chen

2012-03-01

Comparative molecular field analysis (CoMFA) is a widely used 3D-QSAR method by which we can investigate the potential relation between biological activity of compounds and their structural features. In this study, a new application of this approach is presented by combining the molecular modeling with a new developed pharmacophore model specific to CYP1A2 active site. During constructing the model, we used the molecular dynamics simulation and molecular docking method to select the sensible binding conformations for 17 CYP1A2 substrates based on the experimental data. Subsequently, the results obtained via the alignment of binding conformations of substrates were projected onto the active- site residues, upon which a simple blueprint of active site was produced. It was validated by the experimental and computational results that the model did exhibit the high degree of rationality and provide useful insights into the substrate binding. It is anticipated that our approach can be extended to investigate the protein-ligand interactions for many other enzyme-catalyzed systems as well.

Allosteric activation transitions in enzymes and biomolecular motors: insights from atomistic and coarse-grained simulations.

PubMed

Daily, Michael D; Yu, Haibo; Phillips, George N; Cui, Qiang

2013-01-01

The chemical step in enzymes is usually preceded by a kinetically distinct activation step that involves large-scale conformational transitions. In "simple" enzymes this step corresponds to the closure of the active site; in more complex enzymes, such as biomolecular motors, the activation step is more complex and may involve interactions with other biomolecules. These activation transitions are essential to the function of enzymes and perturbations in the scale and/or rate of these transitions are implicated in various serious human diseases; incorporating key flexibilities into engineered enzymes is also considered a major remaining challenge in rational enzyme design. Therefore it is important to understand the underlying mechanism of these transitions. This is a significant challenge to both experimental and computational studies because of the allosteric and multi-scale nature of such transitions. Using our recent studies of two enzyme systems, myosin and adenylate kinase (AK), we discuss how atomistic and coarse-grained simulations can be used to provide insights into the mechanism of activation transitions in realistic systems. Collectively, the results suggest that although many allosteric transitions can be viewed as domain displacements mediated by flexible hinges, there are additional complexities and various deviations. For example, although our studies do not find any evidence for "cracking" in AK, our results do underline the contribution of intra-domain properties (e.g., dihedral flexibility) to the rate of the transition. The study of mechanochemical coupling in myosin highlights that local changes important to chemistry require stabilization from more extensive structural changes; in this sense, more global structural transitions are needed to activate the chemistry in the active site. These discussions further emphasize the importance of better understanding factors that control the degree of co-operativity for allosteric transitions, again

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

PubMed

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Ionizable Side Chains at Catalytic Active Sites of Enzymes

PubMed Central

Jimenez-Morales, David; Liang, Jie

2012-01-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

Discriminative structural approaches for enzyme active-site prediction.

PubMed

Kato, Tsuyoshi; Nagano, Nozomi

2011-02-15

Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

Molecular insight in the multifunctional activities of Withaferin A.

PubMed

Vanden Berghe, Wim; Sabbe, Linde; Kaileh, Mary; Haegeman, Guy; Heyninck, Karen

2012-11-15

Herbal medicine which involves the use of plants for their medicinal value, dates as far back as the origin of mankind and demonstrates an array of applications including cardiovascular protection and anti-cancer activities, via antioxidant, anti-inflammatory and metabolic activities. Even today the popularity of medicinal herbs is still growing like in traditional medicines such as the Indian medicine, Ayurveda. One of the Ayurvedic medicinal plants is Withania somnifera Dunal, of which the important constituents are the withanolides. Among them, Withaferin A is one of the most bioactive compounds, exerting anti-inflammatory, pro-apoptotic but also anti-invasive and anti-angiogenic effects. In the context of modern pharmacology, a better insight in the underlying mechanism of the broad range of bioactivities exerted by Withaferin A is compulsory. Therefore, a lot of effort was made to explore the intracellular effects of Withaferin A and to characterize its target proteins. This review provides a decisive insight on the molecular basis of the health-promoting potential of Withaferin A. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural insight to mutation effects uncover a common allosteric site in class C GPCRs.

PubMed

Harpsøe, Kasper; Boesgaard, Michael W; Munk, Christian; Bräuner-Osborne, Hans; Gloriam, David E

2017-04-15

Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry. Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. We uncover one common site for both positive and negative modulators with different amino acid layouts that can be utilized to obtain selectivity. Additionally, we show a large potential for structure-based modulator design, especially for four orphan receptors with high similarity to the crystal structures. All collated mutagenesis data is available in the GPCRdb mutation browser at http://gpcrdb.org/mutations/ and can be analyzed online or downloaded in excel format. david.gloriam@sund.ku.dk. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦

PubMed Central

Domsic, John F.; Avvaru, Balendu Sankara; Kim, Chae Un; Gruner, Sol M.; Agbandje-McKenna, Mavis; Silverman, David N.; McKenna, Robert

2008-01-01

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. PMID:18768466

Ionizable side chains at catalytic active sites of enzymes.

PubMed

Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

2012-05-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

PubMed Central

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

Metal active site elasticity linked to activation of homocysteine in methionine synthases

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

Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.

2008-04-02

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry uponmore » binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.« less

Human γ-glutamyl transpeptidase 1: Structures of the free enzyme, inhibitor-bound tetrahedral transition states, and glutamate-bound enzyme reveal novel movement within the active site during catalysis [Human gamma-glutamyl transpeptidase: Inhibitor binding and movement within the active site

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

Terzyan, Simon S.; Burgett, Anthony W. G.; Heroux, Annie

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within themore » active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less

Human γ-glutamyl transpeptidase 1: Structures of the free enzyme, inhibitor-bound tetrahedral transition states, and glutamate-bound enzyme reveal novel movement within the active site during catalysis [Human gamma-glutamyl transpeptidase: Inhibitor binding and movement within the active site

DOE PAGES

Terzyan, Simon S.; Burgett, Anthony W. G.; Heroux, Annie; ...

2015-05-26

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within themore » active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less

Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

PubMed

Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

2016-06-17

Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

Quantum mechanical design of enzyme active sites.

PubMed

Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

2008-02-01

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

PubMed Central

Sigala, Paul A.; Fafarman, Aaron T.; Schwans, Jason P.; Fried, Stephen D.; Fenn, Timothy D.; Caaveiro, Jose M. M.; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A.; Boxer, Steven G.; Herschlag, Daniel

2013-01-01

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

PubMed Central

Dunn, Michael F.

2013-01-01

Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR

HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

PubMed Central

Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

2016-01-01

The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll

PubMed Central

Miyazaki, Junichi; Kawagucci, Shinsuke; Makabe, Akiko; Takahashi, Ayu; Kitada, Kazuya; Torimoto, Junji; Matsui, Yohei; Tasumi, Eiji; Shibuya, Takazo; Nakamura, Kentaro; Horai, Shunsuke; Sato, Shun; Ishibashi, Jun-ichiro; Kanzaki, Hayato; Nakagawa, Satoshi; Hirai, Miho; Takaki, Yoshihiro; Okino, Kyoko; Watanabe, Hiromi Kayama; Kumagai, Hidenori

2017-01-01

Since the initial discovery of hydrothermal vents in 1977, these ‘extreme’ chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600–1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed

Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll

NASA Astrophysics Data System (ADS)

Miyazaki, Junichi; Kawagucci, Shinsuke; Makabe, Akiko; Takahashi, Ayu; Kitada, Kazuya; Torimoto, Junji; Matsui, Yohei; Tasumi, Eiji; Shibuya, Takazo; Nakamura, Kentaro; Horai, Shunsuke; Sato, Shun; Ishibashi, Jun-ichiro; Kanzaki, Hayato; Nakagawa, Satoshi; Hirai, Miho; Takaki, Yoshihiro; Okino, Kyoko; Watanabe, Hiromi Kayama; Kumagai, Hidenori; Chen, Chong

2017-12-01

Since the initial discovery of hydrothermal vents in 1977, these `extreme' chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600-1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed.

Active and regulatory sites of cytosolic 5'-nucleotidase.

PubMed

Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

2010-12-01

Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. © 2010 The Authors Journal compilation © 2010 FEBS.

Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

PubMed

Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

2016-09-01

A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

PubMed

Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

1999-03-01

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

Physical activity and cancer risk: dose-response and cancer, all sites and site-specific.

PubMed

Thune, I; Furberg, A S

2001-06-01

The association between physical activity and overall and site-specific cancer risk is elaborated in relation to whether any observed dose-response association between physical activity and cancer can be interpreted in terms of how much physical activity (type, intensity, duration, frequency) is needed to influence site- and gender-specific cancer risk. Observational studies were reviewed that have examined the independent effect of the volume of occupational physical activity (OPA) and/or leisure time physical activity (LPA) on overall and site-specific cancer risk. The evidence of cohort and case-control studies suggests that both leisure time and occupational physical activity protect against overall cancer risk, with a graded dose-response association suggested in both sexes. Confounding effects such as diet, body weight, and parity are often included as a covariate in the analyses, with little influence on the observed associations. A crude graded inverse dose-response association was observed between physical activity and colon cancer in 48 studies including 40,674 colon/colorectal cancer cases for both sexes. A dose-response effect of physical activity on colon cancer risk was especially observed, when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed as MET-hours per week. An observed inverse association with a dose-response relationship between physical activity and breast cancer was also identified in the majority of the 41 studies including 108,031 breast cancer cases. The dose-response relationship was in particular observed in case-control studies and supported by observations in cohort studies when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed by MET-hours per week. This association between physical activity and breast cancer risk is possibly dependent on age at exposure, age at diagnosis, menopausal status and other effect

Neural signatures of attention: insights from decoding population activity patterns.

PubMed

Sapountzis, Panagiotis; Gregoriou, Georgia G

2018-01-01

Understanding brain function and the computations that individual neurons and neuronal ensembles carry out during cognitive functions is one of the biggest challenges in neuroscientific research. To this end, invasive electrophysiological studies have provided important insights by recording the activity of single neurons in behaving animals. To average out noise, responses are typically averaged across repetitions and across neurons that are usually recorded on different days. However, the brain makes decisions on short time scales based on limited exposure to sensory stimulation by interpreting responses of populations of neurons on a moment to moment basis. Recent studies have employed machine-learning algorithms in attention and other cognitive tasks to decode the information content of distributed activity patterns across neuronal ensembles on a single trial basis. Here, we review results from studies that have used pattern-classification decoding approaches to explore the population representation of cognitive functions. These studies have offered significant insights into population coding mechanisms. Moreover, we discuss how such advances can aid the development of cognitive brain-computer interfaces.

Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

PubMed

Miner, Kyle D; Kurtz, Donald M

2016-02-16

HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

Charge density distribution and the electrostatic moments of CTPB in the active site of p300 enzyme: a DFT and charge density study.

PubMed

Devipriya, B; Kumaradhas, P

2013-10-21

A molecular docking and charge density analysis have been carried out to understand the conformational change, charge distribution and electrostatic properties of N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) in the active site of p300. The nearest neighbors, shortest intermolecular contacts between CTPB-p300 and the lowest binding energy of CTPB have been analyzed from the docking analysis. Further, a charge density analysis has been carried out for the molecule in gas phase and for the corresponding molecule lifted from the active site of p300. Due to the intermolecular interaction between CTPB and the amino acids of active site, the conformation of the CTPB has been significantly altered (particularly the pentadecyl chain). CTPB forms strong interaction with the amino acid residues Tyr1397 and Trp1436 at the distance 2.12 and 2.72Å, respectively. However, the long pentadecyl alkyl chain of CTPB produces a barrier and reducing the chance of forming hydrogen bonding with p300. The electron density ρbcp(r) of the polar bonds (C-O, C-N, C-F and C-Cl) of CTPB are increased when it present in the active site. The dipole moment of CTPB in the active site is significantly less (5.73D) when compared with the gas phase (8.16D) form. In the gas phase structure, a large region of negative electrostatic potential (ESP) is found at the vicinity of O(2) and CF3 group, which is less around the O(1) atom. Whereas, in the active site, the negative ESP around the CF3 group is decreased and increased at the O(1) and O(2)-atoms. The ESP modifications of CTPB in the active site are mainly attributed to the effect of intermolecular interaction. The gas phase and active site study insights the molecular flexibility and the electrostatic properties of CTPB in the active site. © 2013 Elsevier Ltd. All rights reserved.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

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

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination ofmore » an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.« less

Understanding the Experience of CACREP On-Site Visiting Review Team Chairpersons

ERIC Educational Resources Information Center

Minor, Amanda J.

2012-01-01

Understanding the experience of CACREP on-site review team members provides insight into the phenomenon of four counselor educators who have each served as a CACREP on-site visiting review team chairperson a minimum of three times. In total, the participants had been within the counselor education field for approximately 95 years and active within…

Diffusional correlations among multiple active sites in a single enzyme.

PubMed

Echeverria, Carlos; Kapral, Raymond

2014-04-07

Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

Black and Brown Bear Activity at Selected Coastal Sites in Glacier Bay National Park and Preserve, Alaska: A Preliminary Assessment Using Noninvasive Procedures

USGS Publications Warehouse

Partridge, Steve; Smith, Tom; Lewis, Tania

2009-01-01

A number of efforts in recent years have sought to predict bear activity in various habitats to minimize human disturbance and bear/human conflicts. Alaskan coastal areas provide important foraging areas for bears (Ursus americanus and U. arctos), particularly following den emergence when there may be no snow-free foraging alternatives. Additionally, coastal areas provide important food items for bears throughout the year. Glacier Bay National Park and Preserve (GLBA) in southeastern Alaska has extensive coastal habitats, and the National Park Service (NPS) has been long interested in learning more about the use of these coastal habitats by bears because these same habitats receive extensive human use by park visitors, especially kayaking recreationists. This study provides insight regarding the nature and intensity of bear activity at selected coastal sites within GLBA. We achieved a clearer understanding of bear/habitat relationships within GLBA by analyzing bear activity data collected with remote cameras, bear sign mapping, scat collections, and genetic analysis of bear hair. Although we could not quantify actual levels of bear activity at study sites, agreement among measures of activity (for example, sign counts, DNA analysis, and video record) lends support to our qualitative site assessments. This work suggests that habitat evaluation, bear sign mapping, and periodic scat counts can provide a useful index of bear activity for sites of interest.

Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

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

Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.

2011-12-22

Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformatemore » inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.« less

A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

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

Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla

2012-04-18

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change ofmore » a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.« less

Toward understanding the active SETI debate: Insights from risk communication and perception

NASA Astrophysics Data System (ADS)

Korbitz, Adam

2014-12-01

Insights from the robust field of risk communication and perception have to date been almost totally absent from the policy debate regarding the relative risks and merits of Active SETI or Messaging to Extraterrestrial Intelligence (METI). For many years, the practice (or proposed practice) of Active SETI has generated a vigorous and sometimes heated policy debate within the scientific community. There have also been some negative reactions in the media toward the activities of those engaged in Active SETI. Risk communication is a scientific approach to communication regarding situations involving potentially sensitive or controversial situations in which there may be high public concern and low public trust. The discipline has found wide acceptance and utility in fields such as public health, industrial regulation and environmental protection. Insights from the scientific field of risk communication (such as omission bias, loss aversion, the availability heuristic, probability neglect, and the general human preference for voluntary over involuntary risks) may help those who have participated in either side of the debate over Active SETI to better understand why the debate has taken on this posture. Principles of risk communication and risk perception may also help those engaged in Active SETI to communicate more effectively with other scientists, the public, with the media, and with policy makers regarding their activities and to better understand and respond to concerns expressed regarding the activity.

Rayleigh Wave Ellipticity Modeling and Inversion for Shallow Structure at the Proposed InSight Landing Site in Elysium Planitia, Mars

NASA Astrophysics Data System (ADS)

Knapmeyer-Endrun, Brigitte; Golombek, Matthew P.; Ohrnberger, Matthias

2017-10-01

The SEIS (Seismic Experiment for Interior Structure) instrument onboard the InSight mission will be the first seismometer directly deployed on the surface of Mars. From studies on the Earth and the Moon, it is well known that site amplification in low-velocity sediments on top of more competent rocks has a strong influence on seismic signals, but can also be used to constrain the subsurface structure. Here we simulate ambient vibration wavefields in a model of the shallow sub-surface at the InSight landing site in Elysium Planitia and demonstrate how the high-frequency Rayleigh wave ellipticity can be extracted from these data and inverted for shallow structure. We find that, depending on model parameters, higher mode ellipticity information can be extracted from single-station data, which significantly reduces uncertainties in inversion. Though the data are most sensitive to properties of the upper-most layer and show a strong trade-off between layer depth and velocity, it is possible to estimate the velocity and thickness of the sub-regolith layer by using reasonable constraints on regolith properties. Model parameters are best constrained if either higher mode data can be used or additional constraints on regolith properties from seismic analysis of the hammer strokes of InSight's heat flow probe HP3 are available. In addition, the Rayleigh wave ellipticity can distinguish between models with a constant regolith velocity and models with a velocity increase in the regolith, information which is difficult to obtain otherwise.

Insight into the fundamental interactions between LEDGF binding site inhibitors and integrase combining docking and molecular dynamics simulations.

PubMed

De Luca, Laura; Morreale, Francesca; Chimirri, Alba

2012-12-21

In recent years, HIV-1 integrase (IN) has emerged as an attractive target for novel anti-AIDS agents. In particular, nonactive-site-binding IN inhibitors would display synergy with current strand-transfer-specific IN inhibitors and other antiretroviral drugs in clinical use. An effective allosteric inhibitory approach would be the disruption of protein-protein interaction (PPI) between IN and cellular cofactors, such as LEDGF/p75. To date, several small molecules have been reported to be inhibitors of the PPI between IN and LEDGF/p75. In this study, we investigated the most relevant interactions between five selected PPI inhibitors and IN comparing them to the naturally occurring IN-LEDGF/p75 complex. We calculated the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA). Total energy was decomposed on per residue contribution, and hydrogen bond occupancies were monitored throughout the simulations. Considering all these results we obtained a good correlation with experimental activity and useful insights for the development of new inhibitors.

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

PubMed Central

Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

2015-01-01

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

PubMed Central

Just, Victoria J.; Burrell, Matthew R.; Bowater, Laura; McRobbie, Iain; Stevenson, Clare E. M.; Lawson, David M.; Bornemann, Stephen

2007-01-01

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural. PMID:17680775

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

High-resolution insight into the competitive adsorption of heavy metals on natural sediment by site energy distribution.

PubMed

Huang, Limin; Jin, Qiang; Tandon, Puja; Li, Aimin; Shan, Aidang; Du, Jiajie

2018-04-01

Investigating competitive adsorption on river/lake sediments is valuable for understanding the fate and transport of heavy metals. Most studies have studied the adsorption isotherms of competitive heavy metals, which mainly comparing the adsorption information on the same concentration. However, intrinsically, the concentration of each heavy metal on competitive adsorption sites is different, while the adsorption energy is identical. Thus, this paper introduced the site energy distribution theory to increase insight into the competitive adsorption of heavy metals (Cu, Cd and Zn). The site energy distributions of each metal with and without other coexisting heavy metals were obtained. It illustrated that site energy distributions provide much more information than adsorption isotherms through screening of the full energy range. The results showed the superior heavy metal in each site energy area and the influence of competitive metals on the site energy distribution of target heavy metal. Site energy distributions can further help in determining the competitive sites and ratios of coexisting metals. In particular, in the high-energy area, which has great environmental significance, the ratios of heavy metals in the competitive adsorption sites obtained for various competitive systems were as follows: slightly more than 3:1 (Cu-Cd), slightly less than 3:1 (Cu-Zn), slightly more than 1:1 (Cd-Zn), and nearly 7:2:2 (Cu-Cd-Zn). The results from this study are helpful to deeply understand competitive adsorption of heavy metals (Cu, Cd, Zn) on sediment. Therefore, this study was effective in presenting a general pattern for future reference in competitive adsorption studies on sediments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

PubMed

Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

2016-05-03

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Uniformity and nonuniformity of neural activities correlated to different insight problem solving.

PubMed

Zhao, Q; Li, Y; Shang, X; Zhou, Z; Han, L

2014-06-13

Previous studies on the neural basis of insight reflected weak consistency except for the anterior cingulate cortex. The present work adopted the semantic and homophonic punny riddle to explore the uniformity and nonuniformity of neural activities correlated to different insight problem solving. Results showed that in the early period of insight solving, the semantic and homophonic punny riddles induced a common N350-500 over the central scalp. However, during -400 to 0 ms before the riddles were solved, the semantic punny riddles induced a positive event-related potential (ERP) deflection over the temporal cortex for retrieving the extensive semantic information, while the homophonic punny riddles induced a positive ERP deflection over the temporal cortex and a negative one in the left frontal cortex which might reflect the semantic and phonological information processing respectively. Our study indicated that different insight problem solving should have the same cognitive process of detecting cognitive conflicts, but have different ways to solve the conflicts. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

PubMed Central

Roosild, Tarmo P.; Castronovo, Samantha

2010-01-01

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications. PMID:20856879

Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

PubMed

Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

2014-07-23

Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

PubMed Central

2015-01-01

Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

The Lusi eruption site: insights from surface and subsurface investigations

NASA Astrophysics Data System (ADS)

Mazzini, A.

2017-12-01

The Indonesian Lusi eruption has been spewing boiling water, gas, and sediments since the 29th of May 2006. Initially, numerous aligned eruptions sites appeared along the Watukosek fault system (WFS) that was reactivated after the Yogyakarta earthquake occurring the 27th of May in the Java Island. Within weeks several villages were submerged by boiling mud. The most prominent eruption site was named Lusi. To date Lusi is still active and an area of 7 km2is covered by mud. Since its birth Lusi erupted with a pulsating behaviour. In the framework of the ERC grant "Lusi Lab" we conducted several years of monitoring and regional investigations coupling surface sampling and subsurface imaging in the region around Lusi. Ambient noise tomography studies, obtained with a local network of 31 stations, revealed for the first time subsurface images of the Lusi region and the adjacent Arjuno-Welirang (AW) volcanic complex. Results show that below the AW volcanic complex are present 5km deep magma chambers that are connected, through a defined corridor, with the roots of the Lusi eruption site. The Lusi subsurface shows the presence of a defined vertical hydrothermal plume that extends to at least 5km. Chemical analyses of the seeping fluids sampled from 1) the Lusi plume (using a specifically designed drone), 2) the region around Lusi, and 3) the fumaroles and the hydro thermal springs of AW, revealed striking similarities. More specifically a mantellic signature of the Lusi fluids confirms the scenario that Lusi represents a magmatic-driven hydrothermal system hosted in sedimentary basin. Seismic profiles interpretation, surface mapping, and fluid sampling show that the WFS, connecting AW and extending towards the NE of Java, acted as a preferential pathway for the igneous intrusion and fluids migration towards the subsurface. Petrography and dating of the clasts erupted at Lusi record high temperatures and indicate that the roots of the active conduit extend to at least 5km

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

PubMed

Jasin, Maria; Haber, James E

2016-08-01

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. Copyright © 2016. Published by Elsevier B.V.

The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

PubMed Central

Jasin, Maria; Haber, James E.

2017-01-01

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. PMID:27261202

Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site.

PubMed

Cao, Nan; Tan, Kemin; Annamalai, Thirunavukkarasu; Joachimiak, Andrzej; Tse-Dinh, Yuk-Ching

2018-06-14

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.

Redefinition of rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues.

PubMed

Kannappan, Babu; Gready, Jill E

2008-11-12

-addition step for residues in three spatially distinct regions of the active site, HIS294 and LYS334 in the C-terminal domain of the large subunit (LSU), but also hitherto unsuspected roles for a cluster of three residues (ASN123, GLU60, and TYR20) in the N-terminal domain of the partner LSU of the dimer containing the active site. Our new mechanism is supported by existing experimental data, provides new convincing interpretations of previously puzzling data, and allows new insights into mutational strategies for improving Rubisco activity.

Addressing Community Concerns about Lead Contamination in Soil: Insights for Site Cleanup

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

Finster, M.E.

2008-07-01

Health risks associated with contaminated sites are a key driver for cleanup decisions and determinations about alternate land use of areas released to the public, particularly in heavily populated metropolitan areas. To guide risk management and future use decisions at contaminated sites, insights can be gained from community-based research. These evaluations can also help ensure that assessments and decisions developed for urban sites consider input received from community members. In order to evaluate the potential risk due to consumption of plants home-grown in lead-contaminated soil, a pilot study was conducted over a period of two summers in a Chicago, ILmore » neighborhood. This survey included analyses of lead concentrations in a convenience sampling of edible fruits, vegetables, and herbs and also examined how the sample preparation method affected the lead concentrations detected in plant materials. A pattern of lead transfer from soil through the root to the stem and leaves of garden crops was found. This pattern is a concern particularly for plants in which the roots, stems, stalks, or leaves are consumed. Analyses of fruiting vegetables indicated that concentrations were below the limit of detection. Depending on the soil lead level and specific plant, the contamination found in some leafy vegetables and herbs may exceed the body's daily excretion rate and could contribute to the total body burden of lead, especially in children. Finally, washing edible portions did not necessarily eliminate the risk, indicating that the lead was located both on and in the plant tissue. This research was conducted in coordination with health experts from the community, and local citizens were involved in discussions on the research and implications for their health protection measures. In certain residential locations, identifying and understanding the potential source of lead contamination provides information for the community such that simple measures can be

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2012 CFR

2012-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2011 CFR

2011-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2013 CFR

2013-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2010 CFR

2010-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2014 CFR

2014-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

Structural insights into the anti-HIV activity of the Oscillatoria agardhii agglutinin homolog lectin family.

PubMed

Koharudin, Leonardus M I; Kollipara, Sireesha; Aiken, Christopher; Gronenborn, Angela M

2012-09-28

Oscillatoria agardhii agglutinin homolog (OAAH) proteins belong to a recently discovered lectin family. All members contain a sequence repeat of ~66 amino acids, with the number of repeats varying among different family members. Apart from data for the founding member OAA, neither three-dimensional structures, information about carbohydrate binding specificities, nor antiviral activity data have been available up to now for any other members of the OAAH family. To elucidate the structural basis for the antiviral mechanism of OAAHs, we determined the crystal structures of Pseudomonas fluorescens and Myxococcus xanthus lectins. Both proteins exhibit the same fold, resembling the founding family member, OAA, with minor differences in loop conformations. Carbohydrate binding studies by NMR and x-ray structures of glycan-lectin complexes reveal that the number of sugar binding sites corresponds to the number of sequence repeats in each protein. As for OAA, tight and specific binding to α3,α6-mannopentaose was observed. All the OAAH proteins described here exhibit potent anti-HIV activity at comparable levels. Altogether, our results provide structural details of the protein-carbohydrate interaction for this novel lectin family and insights into the molecular basis of their HIV inactivation properties.

Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

PubMed

Hylsová, Michaela; Carbain, Benoit; Fanfrlík, Jindřich; Musilová, Lenka; Haldar, Susanta; Köprülüoğlu, Cemal; Ajani, Haresh; Brahmkshatriya, Pathik S; Jorda, Radek; Kryštof, Vladimír; Hobza, Pavel; Echalier, Aude; Paruch, Kamil; Lepšík, Martin

2017-01-27

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R 2  = 0.49). However, the addition of the active-site waters resulted in significant improvement (R 2  = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Design of Conditionally Active STATs: Insights into STAT Activation and Gene Regulatory Function

PubMed Central

Milocco, Lawrence H.; Haslam, Jennifer A.; Rosen, Jonathan; Seidel, H. Martin

1999-01-01

The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity. PMID:10082558

Crystal structures reveal metal-binding plasticity at the metallo-β-lactamase active site of PqqB from Pseudomonas putida

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

Tu, Xiongying; Latham, John A.; Klema, Valerie J.

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone and a distal member of the metallo-β-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here, we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+, and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a widemore » spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal-binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.« less

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

PubMed Central

Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W. H.; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

2010-01-01

Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, 163DVD165 and 221FWGWGREDDE230, are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp224 as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp228 acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics. PMID:20843813

Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

PubMed

Lee, Ciaran M; Davis, Timothy H; Bao, Gang

2018-04-01

What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

PubMed Central

Flanagan, Lindsey A.; Parkin, Alison

2016-01-01

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

Enzyme-ligand interactions that drive active site rearrangements in the Helicobacter pylori 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase

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

Ronning, Donald R; Iacopelli, Natalie M; Mishra, Vidhi

2012-03-15

The bacterial enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays a central role in three essential metabolic pathways in bacteria: methionine salvage, purine salvage, and polyamine biosynthesis. Recently, its role in the pathway that leads to the production of autoinducer II, an important component in quorum-sensing, has garnered much interest. Because of this variety of roles, MTAN is an attractive target for developing new classes of inhibitors that influence bacterial virulence and biofilm formation. To gain insight toward the development of new classes of MTAN inhibitors, the interactions between the Helicobacter pylori-encoded MTAN and its substrates and substrate analogs were probed using X-raymore » crystallography. The structures of MTAN, an MTAN-Formycin A complex, and an adenine bound form were solved by molecular replacement and refined to 1.7, 1.8, and 1.6 Å, respectively. The ribose-binding site in the MTAN and MTAN-adenine cocrystal structures contain a tris[hydroxymethyl]aminomethane molecule that stabilizes the closed form of the enzyme and displaces a nucleophilic water molecule necessary for catalysis. This research gives insight to the interactions between MTAN and bound ligands that promote closing of the enzyme active site and highlights the potential for designing new classes of MTAN inhibitors using a link/grow or ligand assembly development strategy based on the described H. pylori MTAN crystal structures.« less

Structural and mechanistic insights into Mps1 kinase activation.

PubMed

Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

2009-08-01

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-A-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices EF and F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

Structural and mechanistic insights into Mps1 kinase activation

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

Wang, Wei; Yang, Yuting; Gao, Yuefeng

2010-11-05

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation.more » Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.« less

Computer simulation of the active site of human serum cholinesterase

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

Kefang Jiao; Song Li; Zhengzheng Lu

1996-12-31

The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positivemore » electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.« less

Shale gas impacts on groundwater resources: insights from monitoring a fracking site in Poland

NASA Astrophysics Data System (ADS)

Montcoudiol, Nelly; Isherwood, Catherine; Gunning, Andrew; Kelly, Thomas; Younger, Paul

2017-04-01

Exploitation of shale gas by hydraulic fracturing (fracking) is highly controversial and concerns have been raised regarding induced risks from this technique. The SHEER project, an EU Horizon 2020-funded project, is looking into developing best practice to understand, prevent and mitigate the potential short- and long-term environmental impacts and risks from shale gas exploration and exploitation. Three major potential impacts were identified: groundwater contamination, air pollution and induced seismicity. This presentation will deal with the hydrogeological aspect. As part of the SHEER project, four monitoring wells were installed at a shale gas exploration site in Northern Poland. They intercept the main drinking water aquifer located in Quaternary sediments. Baseline monitoring was carried out from mid-December 2015 to beginning of June 2016. Fracking operations occurred in two horizontal wells, in two stages, in June and July 2016. The monitoring has continued after fracking was completed, with site visits every 4-6 weeks. Collected data include measurements of groundwater level, conductivity and temperature at 15-minute intervals, frequent sampling for laboratory analyses and field measurements of groundwater physico-chemical parameters. Groundwater samples are analysed for a range of constituents including dissolved gases and isotopes. The presentation will focus on the interpretation of baseline monitoring data. The insights gained into the behaviour of the Quaternary aquifer will allow a greater perspective to be place on the initial project understanding draw from previous studies. Short-term impacts will also be discussed in comparison with the baseline monitoring results. The presentation will conclude with discussion of challenges regarding monitoring of shale gas fracking sites.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

In situ probing of the active site geometry of ultrathin nanowires for the oxygen reduction reaction

DOE PAGES

Liu, Haiqing; Wong, Stanislaus S.; An, Wei; ...

2015-09-24

To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (~2 nm) core–shell Pt~Pd 9Au nanowires, which have been previouslymore » shown to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu~Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Thus, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general.« less

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

PubMed

Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

2015-04-21

This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

Uncovering Camouflage: Amygdala Activation Predicts Long-Term Memory of Induced Perceptual Insight

PubMed Central

Ludmer, Rachel; Dudai, Yadin; Rubin, Nava

2012-01-01

What brain mechanisms underlie learning of new knowledge from single events? We studied encoding in long-term memory of a unique type of one-shot experience, induced perceptual insight. While undergoing an fMRI brain scan, participants viewed degraded images of real-world pictures where the underlying objects were hard to recognize (‘camouflage’), followed by brief exposures to the original images (‘solution’), which led to induced insight (“Aha!”). A week later, participants’ memory was tested; a solution image was classified as ‘remembered’ if detailed perceptual knowledge was elicited from the camouflage image alone. During encoding, subsequently remembered images enjoyed higher activity in mid-level visual cortex and medial frontal cortex, but most pronouncedly in the amygdala, whose activity could be used to predict which solutions will remain in long-term memory. Our findings extend the known roles of amygdala in memory to include promoting of long-term memory of the sudden reorganization of internal representations. PMID:21382558

Defining the origins of electron transfer at screen-printed graphene-like and graphite electrodes: MoO2 nanowire fabrication on edge plane sites reveals electrochemical insights.

PubMed

Rowley-Neale, Samuel J; Brownson, Dale A C; Banks, Craig E

2016-08-18

Molybdenum (di)oxide (MoO2) nanowires are fabricated onto graphene-like and graphite screen-printed electrodes (SPEs) for the first time, revealing crucial insights into the electrochemical properties of carbon/graphitic based materials. Distinctive patterns observed in the electrochemical process of nanowire decoration show that electron transfer occurs predominantly on edge plane sites when utilising SPEs fabricated/comprised of graphitic materials. Nanowire fabrication along the edge plane sites (and on edge plane like-sites/defects) of graphene/graphite is confirmed with Cyclic Voltammetry, Scanning Electron Microscopy (SEM) and Raman Spectroscopy. Comparison of the heterogeneous electron transfer (HET) rate constants (k°) at unmodified and nanowire coated SPEs show a reduction in the electrochemical reactivity of SPEs when the edge plane sites are effectively blocked/coated with MoO2. Throughout the process, the basal plane sites of the graphene/graphite electrodes remain relatively uncovered; except when the available edge plane sites have been utilised, in which case MoO2 deposition grows from the edge sites covering the entire surface of the electrode. This work clearly illustrates the distinct electron transfer properties of edge and basal plane sites on graphitic materials, indicating favourable electrochemical reactivity at the edge planes in contrast to limited reactivity at the basal plane sites. In addition to providing fundamental insights into the electron transfer properties of graphite and graphene-like SPEs, the reported simple, scalable, and cost effective formation of unique and intriguing MoO2 nanowires realised herein is of significant interest for use in both academic and commercial applications.

Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates.

PubMed

Devine, Erin L; Theobald, Douglas L; Oprian, Daniel D

2016-08-30

The visual pigment rhodopsin is a G protein-coupled receptor that covalently binds its retinal chromophore via a Schiff base linkage to an active-site Lys residue in the seventh transmembrane helix. Although this residue is strictly conserved among all type II retinylidene proteins, we found previously that the active-site Lys in bovine rhodopsin (Lys296) can be moved to three other locations (G90K, T94K, S186K) while retaining the ability to form a pigment with retinal and to activate transducin in a light-dependent manner [ Devine et al. ( 2013 ) Proc. Natl. Acad. Sci. USA 110 , 13351 - 13355 ]. Because the active-site Lys is not functionally constrained to be in helix seven, it is possible that it could relocate within the protein, most likely via an evolutionary intermediate with two active-site Lys. Therefore, in this study we characterized potential evolutionary intermediates with two Lys in the active site. Four mutant rhodopsins were prepared in which the original Lys296 was left untouched and a second Lys residue was substituted for G90K, T94K, S186K, or F293K. All four constructs covalently bind 11-cis-retinal, form a pigment, and activate transducin in a light-dependent manner. These results demonstrate that rhodopsin can tolerate a second Lys in the retinal binding pocket and suggest that an evolutionary intermediate with two Lys could allow migration of the Schiff base Lys to a position other than the observed, highly conserved location in the seventh TM helix. From sequence-based searches, we identified two groups of natural opsins, insect UV cones and neuropsins, that contain Lys residues at two positions in their active sites and also have intriguing spectral similarities to the mutant rhodopsins studied here.

Landscapes, depositional environments and human occupation at Middle Paleolithic open-air sites in the southern Levant, with new insights from Nesher Ramla, Israel

NASA Astrophysics Data System (ADS)

Zaidner, Yossi; Frumkin, Amos; Friesem, David; Tsatskin, Alexander; Shahack-Gross, Ruth

2016-04-01

Middle Paleolithic human occupation in the Levant (250-50 ka ago) has been recorded in roofed (cave and rockshelter) and open-air sites. Research at these different types of sites yielded different perspectives on the Middle Paleolithic human behavior and evolution. Until recently, open-air Middle Paleolithic sites in the Levant were found in three major sedimentary environments: fluvial, lake-margin and spring. Here we describe a unique depositional environment and formation processes at the recently discovered open-air site of Nesher Ramla (Israel) and discuss their contribution to understanding site formation processes in open-air sites in the Levant. The site is 8-m-thick Middle Paleolithic sequence (OSL dated to 170-80 ka) that is located in a karst sinkhole formed by gravitational deformation and sagging into underground voids. The sedimentary sequence was shaped by gravitational collapse, cyclic colluviation of soil and gravel into the depression, waterlogging, in situ pedogenesis and human occupation. Original bedding and combustion features are well-preserved in the Lower archaeological sequence, a rare occurrence in comparison to other open-air archaeological sites. This phenomenon coincides with episodes of fast sedimentation/burial, which also allowed better preservation of microscopic remains such as ash. The Upper archaeological sequence does not exhibit bedding or preservation of ash, despite presence of heat-affected lithic artifacts, which makes it similar to other open-air sites in the Levant. We suggest that rate of burial is the major factor that caused the difference between the Upper and Lower sequences. The differences in the burial rate may be connected to environmental and vegetation changes at the end of MIS 6. We also identified an interplay between sediment in-wash and density of human activity remains, i.e. during episodes of low natural sediment input the density of artifacts is higher relative to episodes with high rate of sediment in

Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

PubMed

Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

2016-08-01

Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes.

PubMed

Zastrow, Melissa L; Pecoraro, Vincent L

2013-04-17

While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

Theoretical Insights to Bulk Activity Towards Oxygen Evolution in Oxyhydroxides

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

Doyle, Andrew D.; Bajdich, Michal; Vojvodic, Aleksandra

The nature of the electrochemical water splitting activity of layered pure and Fe-doped NiOOH is investigated using density functional theory calculations. We find similar thermodynamics for the oxygen evolution reaction (OER) intermediates between the layers of oxyhydroxides, that is, in the bulk of the materials as on the (001) surface. The effect of interlayer spacing on adsorption energy is affected by both the crystal structure and the level of hydrogenation of the active sites. For the Fe-doped NiOOH, we observe general weakening of binding between the different OER intermediates and the catalyst material. The calculated OER activity depends both onmore » doping and interlayer spacing, and our results are generally congruent with available experimental data. In conclusion, these results suggest that such interlayer “bulk” sites may contribute to measured OER activity for both the pure and Fe-doped NiOOH catalysts.« less

Theoretical Insights to Bulk Activity Towards Oxygen Evolution in Oxyhydroxides

DOE PAGES

Doyle, Andrew D.; Bajdich, Michal; Vojvodic, Aleksandra

2017-04-07

The nature of the electrochemical water splitting activity of layered pure and Fe-doped NiOOH is investigated using density functional theory calculations. We find similar thermodynamics for the oxygen evolution reaction (OER) intermediates between the layers of oxyhydroxides, that is, in the bulk of the materials as on the (001) surface. The effect of interlayer spacing on adsorption energy is affected by both the crystal structure and the level of hydrogenation of the active sites. For the Fe-doped NiOOH, we observe general weakening of binding between the different OER intermediates and the catalyst material. The calculated OER activity depends both onmore » doping and interlayer spacing, and our results are generally congruent with available experimental data. In conclusion, these results suggest that such interlayer “bulk” sites may contribute to measured OER activity for both the pure and Fe-doped NiOOH catalysts.« less

Active Sites Environmental Monitoring Program: Mid-FY 1991 report

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

Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

1991-10-01

This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading ofmore » vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.« less

Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

PubMed

Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B; Bach, Ronald R; Nelsestuen, Gary L

2005-04-26

Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

Entrapment and Structure of an Extrahelical Guanine Attempting to Enter the Active Site of a Bacterial DNA Glycosylase, MutM

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

Qi, Yan; Spong, Marie C.; Nam, Kwangho

2010-09-21

MutM, a bacterial DNA glycosylase, protects genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions, thereby initiating base excision DNA repair. The process of searching for and locating oxoG lesions is especially challenging, because of the close structural resemblance of oxoG to its million-fold more abundant progenitor, G. Extrusion of the target nucleobase from the DNA double helix to an extrahelical position is an essential step in lesion recognition and catalysis by MutM. Although the interactions between the extruded oxoG and the active site of MutM have been well characterized, little is known in structural detail regarding themore » interrogation of extruded normal DNA bases by MutM. Here we report the capture and structural elucidation of a complex in which MutM is attempting to present an undamaged G to its active site. The structure of this MutM-extrahelical G complex provides insights into the mechanism MutM employs to discriminate against extrahelical normal DNA bases and into the base extrusion process in general.« less

The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode†

PubMed Central

Caner, Sami; Nguyen, Nham; Aguda, Adeleke; Zhang, Ran; Pan, Yuan T; Withers, Stephen G; Brayer, Gary D

2013-01-01

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation. PMID:23735230

Fragile Sites of 'Valencia' Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA.

PubMed

Lan, Hong; Chen, Chun-Li; Miao, Yin; Yu, Chang-Xiu; Guo, Wen-Wu; Xu, Qiang; Deng, Xiu-Xin

2016-01-01

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.

All the catalytic active sites of MoS 2 for hydrogen evolution

DOE PAGES

Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

2016-11-29

MoS 2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS 2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated tomore » be 7.5 s –1 (65–75 mV/dec), 3.2 s –1 (65–85 mV/dec), and 0.1 s –1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS 2 is higher than that in low crystalline quality MoS 2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

New insights into the molecular characteristics behind the function of Renilla luciferase.

PubMed

Fanaei-Kahrani, Zahra; Ganjalikhany, Mohamad Reza; Rasa, Seyed Mohammad Mahdi; Emamzadeh, Rahman

2018-02-01

Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8. © 2017 Wiley Periodicals, Inc.

Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

DOE PAGES

Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping; ...

2017-07-27

Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

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

Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping

Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity.

PubMed

Chen, Zhi-Wei; Datta, Saumen; Dubois, Jennifer L; Klinman, Judith P; Mathews, F Scott

2010-08-31

The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies

NASA Astrophysics Data System (ADS)

Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

2011-06-01

The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies.

PubMed

Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

2011-06-01

The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

Insights on activation enthalpy for non-Schmid slip in body-centered cubic metals

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

Hale, Lucas M.; Lim, Hojun; Zimmerman, Jonathan A.

2014-12-18

We use insights gained from atomistic simulation to develop an activation enthalpy model for dislocation slip in body-centered cubic iron. Furthermore, using a classical potential that predicts dislocation core stabilities consistent with ab initio predictions, we quantify the non-Schmid stress-dependent effects of slip. The kink-pair activation enthalpy is evaluated and a model is identified as a function of the general stress state. Thus, our model enlarges the applicability of the classic Kocks activation enthalpy model to materials with non-Schmid behavior.

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Chapter 12: Daily Patterns of Marbled Murrelet Activity at Inland Sites

Treesearch

Nancy L. Naslund; Brian P. O’Donnell

1995-01-01

Patterns in the daily activity of Marbled Murrelets (Brachyramphus marmoratus) at inland sites has been studied throughout their range from California to Alaska. Murrelets are most active at inland sites around dawn, and to a lesser degree, at dusk. Throughout their range, peak levels of activity (detections) occur in the hour around dawn, but...

An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

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

Eren, Elif; Murphy, Megan; Goguen, Jon

2010-08-13

The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changesmore » of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.« less

Proofs that Develop Insight

ERIC Educational Resources Information Center

Weber, Keith

2010-01-01

Many mathematics educators have noted that mathematicians do not only read proofs to gain conviction but also to obtain insight. The goal of this article is to discuss what this insight is from mathematicians' perspective. Based on interviews with nine research-active mathematicians, two sources of insight are discussed. The first is reading a…

Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

PubMed Central

2015-01-01

Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

NASA Astrophysics Data System (ADS)

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-01

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

PubMed

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-05

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. Copyright © 2016 Elsevier B.V. All rights reserved.

Conformational Changes Allow Processing of Bulky Substrates by a Haloalkane Dehalogenase with a Small and Buried Active Site.

PubMed

Kokkonen, Piia; Bednar, David; Dockalova, Veronika; Prokop, Zbynek; Damborsky, Jiri

2018-06-01

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and which is likely the route for bulky substrates. These results provide insights into the structure-dynamics-function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to DCE introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

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

Wang, Hui; Klein, Michael G.; Snell, Gyorgy

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structuremore » reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.« less

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

Observing the formation of ice and organic crystals in active sites

PubMed Central

Campbell, James M.; Meldrum, Fiona C.; Christenson, Hugo K.

2017-01-01

Heterogeneous nucleation is vital to a wide range of areas as diverse as ice nucleation on atmospheric aerosols and the fabrication of high-performance thin films. There is excellent evidence that surface topography is a key factor in directing crystallization in real systems; however, the mechanisms by which nanoscale pits and pores promote nucleation remain unclear. Here, we use natural cleavage defects on Muscovite mica to investigate the activity of topographical features in the nucleation from vapor of ice and various organic crystals. Direct observation of crystallization within surface pockets using optical microscopy and also interferometry demonstrates that these sharply acute features provide extremely effective nucleation sites and allows us to determine the mechanism by which this occurs. A confined phase is first seen to form along the apex of the wedge and then grows out of the pocket opening to generate a bulk crystal after a threshold saturation has been achieved. Ice nucleation proceeds in a comparable manner, although our resolution is insufficient to directly observe a condensate before the growth of a bulk crystal. These results provide insight into the mechanism of crystal deposition from vapor on real surfaces, where this will ultimately enable us to use topography to control crystal deposition on surfaces. They are also particularly relevant to our understanding of processes such as cirrus cloud formation, where such topographical features are likely candidates for the “active sites” that make clay particles effective nucleants for ice in the atmosphere. PMID:27994140

Feeling bogged down about climate change mitigation? Insights from a new high resolution peatland-bog model validated at two Dutch monitoring sites.

NASA Astrophysics Data System (ADS)

Lippmann, Tanya; van Huissteden, Ko; Hendriks, Dimmie

2017-04-01

Increasing the global carbon sink is one of two options to mitigate CO2 and CH4 increases in the atmosphere (the other is emissions reductions at the source). Peatlands release carbon to the atmosphere when disturbed by natural or human causes and absorb carbon when vegetation and soil organic matter accumulate after rewetting or natural revegetation. However, rewetting of drained peatlands is frequently not considered as a climate mitigation strategy due to the enhanced methane emissions that accompany newly formed anaerobic peatland environments. We hypothesise that at most sites, this trend will be temporal but long-term, lasting for tens of years post re-wetting before stabilisation takes place. This study investigates the ability of rewetted peatland sites to act as either a source or sink for atmospheric methane and carbon dioxide under climate change. The hydrology of a peatland is fundamental to its functioning. Therefore, the use of a full water balance table has the potential to simulate greenhouse gas fluxes to a greater degree of certainty. MODFLOW is the internationally most widely used ground and surface water model and is freely available to the scientific community. This is the first time that a gridded peatland process based model has been constructed at a spatial resolution as high as 25m x 25m. This new high-resolution model allows for investigation into the complex biophysical and hydrological factors that are necessary to reliably estimate atmospheric greenhouse gas fluxes in a peatland ecosystem. We assess the model's skill against observations collected at two monitoring sites of differing soil properties and vegetation in the Netherlands. These results discuss site-specific suitability of peatland regeneration, useful for climate change mitigation activities. Aside from the insight into transient atmosphere-peatland carbon fluxes, this work is a stepping stone towards more robust model coupling and greater spatial coverage.

Active-site solvent replenishment observed during human carbonic anhydrase II catalysis.

PubMed

Kim, Jin Kyun; Lomelino, Carrie L; Avvaru, Balendu Sankara; Mahon, Brian P; McKenna, Robert; Park, SangYoun; Kim, Chae Un

2018-01-01

Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO 2 /HCO 3 - . Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO 2 pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (W I ') is observed next to the previously observed intermediate water W I , and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH - on CO 2 . This mechanism explains how the zinc-bound water (W Zn ) and W1 are replenished, which are directly responsible for the reconnection of the His64-mediated proton-transfer water network. This study provides the first 'physical' glimpse of how a water reservoir flows into the hCA II active site during its catalytic activity.

Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase*

PubMed Central

Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

2012-01-01

Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases. PMID:22356908

Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites.

PubMed

Ribeiro, António J M; Holliday, Gemma L; Furnham, Nicholas; Tyzack, Jonathan D; Ferris, Katherine; Thornton, Janet M

2018-01-04

M-CSA (Mechanism and Catalytic Site Atlas) is a database of enzyme active sites and reaction mechanisms that can be accessed at www.ebi.ac.uk/thornton-srv/m-csa. Our objectives with M-CSA are to provide an open data resource for the community to browse known enzyme reaction mechanisms and catalytic sites, and to use the dataset to understand enzyme function and evolution. M-CSA results from the merging of two existing databases, MACiE (Mechanism, Annotation and Classification in Enzymes), a database of enzyme mechanisms, and CSA (Catalytic Site Atlas), a database of catalytic sites of enzymes. We are releasing M-CSA as a new website and underlying database architecture. At the moment, M-CSA contains 961 entries, 423 of these with detailed mechanism information, and 538 with information on the catalytic site residues only. In total, these cover 81% (195/241) of third level EC numbers with a PDB structure, and 30% (840/2793) of fourth level EC numbers with a PDB structure, out of 6028 in total. By searching for close homologues, we are able to extend M-CSA coverage of PDB and UniProtKB to 51 993 structures and to over five million sequences, respectively, of which about 40% and 30% have a conserved active site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Theoretical Insights into Methane C–H Bond Activation on Alkaline Metal Oxides

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

Aljama, Hassan; Nørskov, Jens K.; Abild-Pedersen, Frank

Here, we investigate the role of alkaline metal oxides (AMO) (MgO, CaO, and SrO) in activating the C–H bond in methane. We also use Density Functional Theory (DFT) and microkinetic modeling to study the catalytic elementary steps in breaking the C–H bond in methane and creating the methyl radical, a precursor prior to creating C2 products. We also study the effects of surface geometry on the catalytic activity of AMO by examining terrace and step sites. We observe that the process of activating methane depends strongly on the structure of the AMO. When the AMO surface is doped with anmore » alkali metal, the transition state (TS) structure has a methyl radical-like behavior, where the methyl radical interacts weakly with the AMO surface. In this case, the TS energy scales with the hydrogen binding energy. On pure AMO, the TS interacts with AMO surface oxygen as well as the metal atom on the surface, and consequently the TS energy scales with the binding energy of hydrogen and methyl. We study the activity of AMO using a mean-field microkinetic model. The results indicate that terrace sites have similar catalytic activity, with the exception of MgO(100). Step sites bind hydrogen more strongly, making them more active, and this confirms previously reported experimental results. We map the catalytic activity of AMO using a volcano plot with two descriptors: the methyl and the hydrogen binding energies, with the latter being a more significant descriptor. The microkinetic model results suggest that C–H bond dissociation is not always the rate-limiting step. At weak hydrogen binding, the reaction is limited by C–H bond activation. At strong hydrogen binding, the reaction is limited due to poisoning of the active site. We found an increase in activity of AMO as the basicity increased. Finally, the developed microkinetic model allows screening for improved catalysts using simple calculations of the hydrogen binding energy.« less

Theoretical Insights into Methane C–H Bond Activation on Alkaline Metal Oxides

DOE PAGES

Aljama, Hassan; Nørskov, Jens K.; Abild-Pedersen, Frank

2017-07-17

Here, we investigate the role of alkaline metal oxides (AMO) (MgO, CaO, and SrO) in activating the C–H bond in methane. We also use Density Functional Theory (DFT) and microkinetic modeling to study the catalytic elementary steps in breaking the C–H bond in methane and creating the methyl radical, a precursor prior to creating C2 products. We also study the effects of surface geometry on the catalytic activity of AMO by examining terrace and step sites. We observe that the process of activating methane depends strongly on the structure of the AMO. When the AMO surface is doped with anmore » alkali metal, the transition state (TS) structure has a methyl radical-like behavior, where the methyl radical interacts weakly with the AMO surface. In this case, the TS energy scales with the hydrogen binding energy. On pure AMO, the TS interacts with AMO surface oxygen as well as the metal atom on the surface, and consequently the TS energy scales with the binding energy of hydrogen and methyl. We study the activity of AMO using a mean-field microkinetic model. The results indicate that terrace sites have similar catalytic activity, with the exception of MgO(100). Step sites bind hydrogen more strongly, making them more active, and this confirms previously reported experimental results. We map the catalytic activity of AMO using a volcano plot with two descriptors: the methyl and the hydrogen binding energies, with the latter being a more significant descriptor. The microkinetic model results suggest that C–H bond dissociation is not always the rate-limiting step. At weak hydrogen binding, the reaction is limited by C–H bond activation. At strong hydrogen binding, the reaction is limited due to poisoning of the active site. We found an increase in activity of AMO as the basicity increased. Finally, the developed microkinetic model allows screening for improved catalysts using simple calculations of the hydrogen binding energy.« less

Fragile Sites of ‘Valencia’ Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA

PubMed Central

Lan, Hong; Chen, Chun-Li; Miao, Yin; Yu, Chang-Xiu; Guo, Wen-Wu; Xu, Qiang; Deng, Xiu-Xin

2016-01-01

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in ‘Valencia’ C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of ‘Valencia’ C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid ‘Valencia’ C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in ‘Valencia’ sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in ‘Valencia’ sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites. PMID:26977938

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

PubMed

Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

2018-05-03

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

SABER: A computational method for identifying active sites for new reactions

PubMed Central

Nosrati, Geoffrey R; Houk, K N

2012-01-01

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were l-Ala d/l-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

SABER: a computational method for identifying active sites for new reactions.

PubMed

Nosrati, Geoffrey R; Houk, K N

2012-05-01

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. Copyright © 2012 The Protein Society.

'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

PubMed

Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

2014-04-09

The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

PubMed

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

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

Lau, E Y; Felton, J S; Lightstone, F C

2006-06-06

A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclicmore » amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.« less

Facebook, Twitter Activities Sites, Location and Students' Interest in Learning

ERIC Educational Resources Information Center

Igbo, J. N.; Ezenwaji, Ifeyinwa; Ajuziogu, Christiana U.

2018-01-01

This study was carried out to ascertain the influence of social networking sites activities (twitter and Facebook) on secondary school students' interest in learning It also considered the impact of these social networking sites activities on location of the students. Two research questions and two null hypotheses guided the study. Mean and…

Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

PubMed Central

2016-01-01

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

PubMed

Blaha-Nelson, David; Krüger, Dennis M; Szeler, Klaudia; Ben-David, Moshe; Kamerlin, Shina Caroline Lynn

2017-01-25

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the k cat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.

Characterization of the active site properties of CYP4F12.

PubMed

Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

2014-10-01

Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Dust devil track survey at Elysium Planitia, Mars: Implications for the InSight landing sites

NASA Astrophysics Data System (ADS)

Reiss, Dennis; Lorenz, Ralph D.

2016-03-01

The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) robotic lander is scheduled to land in Elysium Planitia on Mars in September 2016. InSight will perform the first comprehensive surface-based geophysical investigation including seismic measurements. Knowledge about encounter rates of dust devils with the InSight lander are important for two main reasons: (1) dust devils will affect the scientific measurements, i.e., wind-induced seismic noise, and (2) the power-supply of the InSight lander and instruments is provided by solar arrays and previous landers and rovers on Mars were affected by a steady decline in electrical power output due to atmospheric dust deposition on the solar panels. Long term science operations were only made possible by dust clearing events of the solar arrays caused by wind gusts and dust devils. In this study we analyzed dust devil tracks (DDTs) at the final InSight landing site region in Elysium Planitia. Formation of DDTs is caused by the removal of a layer of dust by passing dust devils, hence in principle the same process as clearing of dust from solar panels. We mapped the number, size (width and length), and orientation of DDTs in repeat observations using High Resolution Imaging Science Experiment (HiRISE) images covering the exact same surface area acquired within a relatively short time span (<90 martian days). In total, we analyzed 557 newly formed dust devil tracks in 8 study areas. DDTs are morphologically relatively straight with a low mean sinuosity of 1.03 and only reach maximum widths of 30 m. The mean DDT width is 4 m, indicating that the dust devil size population is dominated by small dust devils with a diameter <10 m. The size-frequency distribution of DDTs follows a -2 power law. The mean lengths of DDTs are 0.62 km and 1.23 km for complete (tracks which are visible from their start to end point) and incomplete DDTs (tracks running across the HiRISE footprint), respectively. The

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

PubMed

Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

2016-11-01

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

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

Gopal, B.; Madan, Lalima L.; Betz, Stephen F.

2010-11-10

Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s),more » whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.« less

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

PubMed Central

He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Chan, Edward Wai-Chi

2016-01-01

Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity. PMID:27480856

Deprotonation states of the two active site water molecules regulate the binding of protein phosphatase 5 with its substrate: A molecular dynamics study.

PubMed

Wang, Lingyun; Yan, Feng

2017-10-01

Protein phosphatase 5 (PP5), mainly localized in human brain, can dephosphorylate tau protein whose high level of phosphorylation is related to Alzheimer's disease. Similar to other protein phosphatases, PP5 has a conserved motif in the catalytic domain that contains two binding sites for manganese (Mn 2+ ) ions. Structural data indicate that two active site water molecules, one bridging the two Mn 2+ ions and the other terminally coordinated with one of the Mn 2+ ions (Mn1), are involved in catalysis. Recently, a density functional theory study revealed that the two water molecules can be both deprotonated to keep a neutral active site for catalysis. The theoretical study gives us an insight into the catalytic mechanism of PP5, but the knowledge of how the deprotonation states of the two water molecules affect the binding of PP5 with its substrate is still lacking. To approach this problem, molecular dynamics simulations were performed to model the four possible deprotonation states. Through structural, dynamical and energetic analyses, the results demonstrate that the deprotonation states of the two water molecules affect the structure of the active site including the distance between the two Mn 2+ ions and their coordination, impact the interaction energy of residues R275, R400 and H304 which directly interact with the substrate phosphoserine, and mediate the dynamics of helix αJ which is involved in regulation of the enzyme's activity. Furthermore, the deprotonation state that is preferable for PP5 binding of its substrate has been identified. These findings could provide new design strategy for PP5 inhibitor. © 2017 The Protein Society.

A hybrid QM/MM simulation study of intramolecular proton transfer in the pyridoxal 5'-phosphate in the active site of transaminase: influence of active site interaction on proton transfer.

PubMed

Dutta Banik, Sindrila; Chandra, Amalendu

2014-09-25

Pyridoxal 5'-phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium that involves an intramolecular proton transfer between the N-protonated zwitterionic ketoenamine tautomer and the O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in the zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP aspartate (PLP-Asp) Schiff base in the active site of aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical simulations. The main focus of the present study is to analyze the factors that control the tautomeric equilibrium in the active sites of various PLP-dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for the ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in the ketoenamine tautomer, while in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP-dependent enzymes (aspartate aminotransferase, Dopa decarboxylase, and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium toward the N

Community Update on Site Activities, July 19, 2013

EPA Pesticide Factsheets

In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

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

Crichlow, G.; Lubetsky, J; Leng, L

Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic datamore » indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.« less

Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites.

PubMed

Chen, Baoyu; Chou, Hui-Ting; Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas; Rosen, Michael K

2017-09-26

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

DOE PAGES

Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

2016-08-17

Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [Cu IIOH] + species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [Cu IIOH] + active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu] 2+ and Cu 3O 3 motifs.« less

What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

PubMed

Zhang, Ni; Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

2017-06-22

Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. ©Ni Zhang, Shelly Campo, Jingzhen Yang, Petya Eckler, Linda Snetselaar, Kathleen Janz, Emily Leary. Originally published in the Journal of Medical

What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

PubMed Central

Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

2017-01-01

Background Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. Objective This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. Methods A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. Results The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. Conclusions This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. PMID:28642215

Corrosion Research And Web Site Activities

NASA Technical Reports Server (NTRS)

Heidersbach, Robert H.

2001-01-01

This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

Corrosion Research and Web Site Activities

NASA Technical Reports Server (NTRS)

Heidersbach, Robert H.

2002-01-01

This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics.

PubMed

Bauler, Patricia; Huber, Gary; Leyh, Thomas; McCammon, J Andrew

2010-05-06

Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.

Effect of Siloxane Ring Strain and Cation Charge Density on the Formation of Coordinately Unsaturated Metal Sites on Silica: Insights from Density Functional Theory (DFT) Studies

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

Das, Ujjal; Zhang, Guanghui; Hu, Bo

2015-10-28

Amorphous silica (SiO 2) is commonly used as a support in heterogeneous catalysis. However, due to the structural disorder and temperature induced change of surface morphology, the structures of silica supported metal catalysts are difficult to determine. Most studies are primarily focused on understanding the interactions of different types of surface hydroxyl groups with metal ions. In comparison, the effect of siloxane ring size on the structure of silica supported metal catalysts and how it affects catalytic activity is poorly understood. Here, we have used density functional theory calculations to understand the effect of siloxane ring strain on structure andmore » activity of different monomeric Lewis acid metal sites on silica. In particular, we have found that large siloxane rings favor strong dative bonding interaction between metal ion and surface hydroxyls, leading to the formation of high-coordinate metal sites. In comparison, metal-silanol interaction is weak in small siloxane rings, resulting in low-coordinate metal sites. The physical origin of this size dependence is associated with siloxane ring strain, and, a correlation between metal-silanol interaction energy and ring strain energy has been observed. In addition to ring strain, the strength of the metal-silanol interaction also depends on the positive charge density of the cations. In fact, a correlation also exists between metal-silanol interaction energy and charge density of several first-row transition and post-transition metals. The theoretical results are compared with the EXAFS data of monomeric Zn(II) and Ga(III) ions grafted on silica. In conclusion, the molecular level insights of how metal ion coordination on silica depends on siloxane ring strain and cation charge density will be useful in the synthesis of new catalysts.« less

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

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

Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben

2017-03-28

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by themore » SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.« less

Shallow structure of the InSight 2018 landing site in Elysium Planitia, Mars, from ambient vibration Rayleigh wave ellipticity: A modeling study

NASA Astrophysics Data System (ADS)

Knapmeyer-Endrun, B.; Golombek, M.; Ohrnberger, M. M.

2016-12-01

The SEIS (Seismic Experiment for Interior Structure) instrument onboard NASA's InSight mission, scheduled to land in November 2018, will be the first seismometer directly deployed on the surface of Mars. From studies on both the Earth and the Moon, it is well known that site amplification in low-velocity sediments, e.g. regolith, on top of more competent rocks has a strong influence on seismic signals, but can also be used to constrain the subsurface structure. Based on orbital data, lab measurements and terrestrial analogues, we construct a model of the shallow sub-surface at the landing site in western Elysium Planitia and simulate the ambient vibration wavefield. We show how Rayleigh wave ellipticity can be extracted from these data and inverted for shallow structure. Using reasonable variations in regolith properties, we do not expect any influence of site resonances on teleseismic quakes recorded by InSight, but recordings of local events will likely be affected. We find that higher mode ellipticity information might be extracted from the data, significantly reducing uncertainties in the inversion. Though the data are most sensitive to properties of the upper-most layer and show a strong trade-off between layer depth and velocity, it is possible to estimate the velocity and thickness of the sub-regolith layer and distinguish between different models by using reasonable constraints on regolith properties. Model parameters are best constrained if either higher mode data can be used or additional constraints on regolith properties, e.g. from analysis of hammer strokes of the HP3 heat flow probe or orbital mapping of regolith thickness from the onset diameter of rocky ejecta craters, are available. In addition, Rayleigh wave ellipticity can differentiate between models with a constant regolith velocity and models with increasing velocity with depth. We also discuss the influence of lander and leveling system mechanical noise on the identification of site

Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

PubMed

Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

2018-02-14

Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

Pyrrole-Based Antitubulin Agents: Two Distinct Binding Modalities are Predicted for C-2 Analogs in the Colchicine Site.

PubMed

Da, Chenxiao; Telang, Nakul; Barelli, Peter; Jia, Xin; Gupton, John T; Mooberry, Susan L; Kellogg, Glen E

2012-01-12

3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogs were synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct binding modes that explain the structural-activity relationships and are in accord with the structural basis of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the colchicine site and compound design.

Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts

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

Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

The activation of molecular O 2as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O 2activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O 2dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O 2dissociationmore » is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O 2dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction.« less

Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts

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

Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

2016-01-01

The activation of molecular O 2as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O 2activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O 2dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O 2dissociationmore » is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O 2dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction.« less

Brownian aggregation rate of colloid particles with several active sites

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

Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V., E-mail: chern@ns.kinetics.nsc.ru

2014-08-14

We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shownmore » to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.« less

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

PubMed

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

Reversible active site sulfoxygenation can explain the oxygen tolerance of a NAD+-reducing [NiFe] hydrogenase and its unusual infrared spectroscopic properties.

PubMed

Horch, Marius; Lauterbach, Lars; Mroginski, Maria Andrea; Hildebrandt, Peter; Lenz, Oliver; Zebger, Ingo

2015-02-25

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD(+)-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD(+) and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2.

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.

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes.

PubMed

He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Chan, Edward Wai-Chi; Liu, Jian-Hua; Chen, Sheng

2016-10-01

Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

PubMed

Chowdhury, Sandipan; Chanda, Baron

2010-11-02

Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

PubMed Central

Treuheit, Nicholas A.; Beach, Muneera A.; Komives, Elizabeth A.

2011-01-01

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethylketone to the active site serine, as well as non-covalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1, however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-L-arginine-(3-methyl-1,5-pantanediyl) amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause the same reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or to exosite 1. PMID:21526769

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

PubMed

Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

2011-05-31

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

Passive colloids work together to become Active

NASA Astrophysics Data System (ADS)

Kandula, Hima Nagamanasa; Wang, Wei; Zhang, Jie; Wu, Huanxin; Han, Ming; Luijten, Erik; Granick, Steve

In recent years there is growing body of research to design self-propelled colloids to gain insights into non-equilibrium systems including living matter. While most active colloids developed hitherto entail prefabrication of Janus colloids and possess single fixed active site, we present one simple system where active colloids are formed in-situ naturally with multiple active sites and are reversible as well as reconfigurable. A binary mixture of Brownian colloids which have opposite polarizations when subjected to an AC electric field spontaneously assemble into clusters which are propelled by asymmetric induced charge electro osmosis. We find that tuning the relative sizes of the two species allows for the control over the number of active sites. More interestingly, the patches are dynamic enabling reconfiguration of the active cluster. Consequently, the clusters are active not only in motion but also in their structure.

Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

PubMed Central

Miao, Yinglong; Baudry, Jerome

2011-01-01

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site. PMID:21943431

Active Site Mutations Change the Cleavage Specificity of Neprilysin

PubMed Central

Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

2012-01-01

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana.

PubMed

Betsuyaku, Shigeyuki; Katou, Shinpei; Takebayashi, Yumiko; Sakakibara, Hitoshi; Nomura, Nobuhiko; Fukuda, Hiroo

2018-01-01

The innate immune response is, in the first place, elicited at the site of infection. Thus, the host response can be different among the infected cells and the cells surrounding them. Effector-triggered immunity (ETI), a form of innate immunity in plants, is triggered by specific recognition between pathogen effectors and their corresponding plant cytosolic immune receptors, resulting in rapid localized cell death known as hypersensitive response (HR). HR cell death is usually limited to a few cells at the infection site, and is surrounded by a few layers of cells massively expressing defense genes such as Pathogenesis-Related Gene 1 (PR1). This virtually concentric pattern of the cellular responses in ETI is proposed to be regulated by a concentration gradient of salicylic acid (SA), a phytohormone accumulated around the infection site. Recent studies demonstrated that jasmonic acid (JA), another phytohormone known to be mutually antagonistic to SA in many cases, is also accumulated in and required for ETI, suggesting that ETI is a unique case. However, the molecular basis for this uniqueness remained largely to be solved. Here, we found that, using intravital time-lapse imaging, the JA signaling pathway is activated in the cells surrounding the central SA-active cells around the infection sites in Arabidopsis thaliana. This distinct spatial organization explains how these two phythormone pathways in a mutually antagonistic relationship can be activated simultaneously during ETI. Our results re-emphasize that the spatial consideration is a key strategy to gain mechanistic insights into the apparently complex signaling cross-talk in immunity. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Carbinolamine Formation and Dehydration in a DNA Repair Enzyme Active Site

PubMed Central

Dodson, M. L.; Walker, Ross C.; Lloyd, R. Stephen

2012-01-01

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics–molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water–independent enzyme–catalyzed reaction had a bias–corrected Jarzynski–average barrier height of approximately for the carbinolamine formation reaction and ) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately in the forward (formation) reaction and for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water–independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N–terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site

Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

PubMed

Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

2014-06-01

Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis.

PubMed

Perez, Rodney H; Sugino, Haruki; Ishibashi, Naoki; Zendo, Takeshi; Wilaipun, Pongtep; Leelawatcharamas, Vichien; Nakayama, Jiro; Sonomoto, Kenji

2017-04-01

Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

The Ecosystem-Economy Relationship: Insights from Six Forested LTER Sites

Treesearch

Paul N. Courant; Ernie Niemi; Ed. Whitelaw

1997-01-01

The debate over forest-management policy in the U.S. often is cast as a choice between jobs and [pick the environmental attribute of your choice]. The purpose of this paper is neither to rehash nor to characterize these conflicts, but to discuss insights into them that have emerged from an examination of the forest-economy relationship in different regions of the U.S....

InSight: An innovative multimedia training tool

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

Seidel, B.R.; Crites, D.C.; Forsmann, J.H.

1996-05-01

InSight is an innovative computer-based multimedia training tool that provides a navigable virtual environment and links to related information. It provides training and guidance for touring and observing operations at any facility or site in a realistic virtual environment. This presentation identifies unique attributes of InSight and describes the initial application at ANL-West. A brief description of the development of this tool, production steps, and an onscreen demonstration of its operation are also provided.

Ultrafast Light-Driven Substrate Expulsion from the Active Site of a Photoswitchable Catalyst.

PubMed

Pescher, Manuel D; van Wilderen, Luuk J G W; Grützner, Susanne; Slavov, Chavdar; Wachtveitl, Josef; Hecht, Stefan; Bredenbeck, Jens

2017-09-25

The photoswitchable piperidine general base catalyst is a prototype structure for light control of catalysis. Its azobenzene moiety moves sterically shielding groups to either protect or expose the active site, thereby changing the basicity and hydrogen-bonding affinity of the compound. The reversible switching dynamics of the catalyst is probed in the infrared spectral range by monitoring hydrogen bond (HB) formation between its active site and methanol (MeOH) as HB donor. Steady-state infrared (IR) and ultrafast IR and UV/Vis spectroscopies are used to uncover ultrafast expulsion of MeOH from the active site within a few picoseconds. Thus, the force generated by the azobenzene moiety even in the final phase of its isomerization is sufficient to break a strong HB within 3 ps and to shut down access to the active site. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insight in schizophrenia: involvement of self-reflection networks?

PubMed

van der Meer, Lisette; de Vos, Annerieke E; Stiekema, Annemarie P M; Pijnenborg, Gerdina H M; van Tol, Marie-José; Nolen, Willem A; David, Anthony S; Aleman, André

2013-11-01

Impaired insight is a common feature in psychosis and an important predictor of variables such as functional outcome, prognosis, and treatment adherence. A cognitive process that may underlie insight in psychosis is self-reflection, or the conscious evaluation of one's traits and characteristics. The current study aims to investigate the neural correlates of self-reflective processing and its relationship with insight in schizophrenia. Forty-seven schizophrenia patients and 21 healthy controls performed a self-reflection task in a functional magnetic resonance imaging (fMRI) scanner. The tasks comprised a self-reflection, close other-reflection, and a semantic (baseline) condition. Insight scores were obtained with the Schedule of Assessment of Insight Expanded. In addition, cognitive insight scores were obtained (Beck Cognitive Insight Scale [BCIS]). Schizophrenia patients demonstrated less activation in the posterior cingulate cortex in the self- and other-reflection conditions and less activation in the precuneus in the other-reflection condition compared with healthy controls. Better insight was associated with greater response in the inferior frontal gyrus, anterior insula, and inferior parietal lobule during self-reflection. In addition, better cognitive insight was associated with higher activation in ventromedial prefrontal cortex during self-reflection. In the current study, evidence for a relationship between self-reflection and insight in patients with schizophrenia was found in brain areas related to self-reflection, self/other distinction and source attribution. The findings support the rationale for a treatment that is currently under evaluation, which attempts to increase insight by enhancing self-reflection.

Landing Area Narrowed for 2016 InSight Mission to Mars

NASA Image and Video Library

2013-09-04

The process of selecting a site for NASA's next landing on Mars, planned for September 2016, has narrowed to four semifinalist sites located close together in the Elysium Planitia region of Mars. The mission known by the acronym InSight will study the Red Planet's interior, rather than surface features, to advance understanding of the processes that formed and shaped the rocky planets of the inner solar system, including Earth. The location of the cluster of semifinalist landing sites for InSight is indicated on this near-global topographic map of Mars, which also indicates landing sites of current and past NASA missions to the surface of Mars. The mission's full name is Interior Exploration Using Seismic Investigations, Geodesy and Heat Transport. The location of Elysium Planitia close to the Martian equator meets an engineering requirement for the stationary InSight lander to receive adequate solar irradiation year-round on its photovoltaic array. The location also meets an engineering constraint for low elevation, optimizing the amount of atmosphere the spacecraft can use for deceleration during its descent to the surface. The number of candidate landing sites for InSight was trimmed from 22 down to four in August 2013. This down-selection facilitates focusing the efforts to further evaluate the four sites. Cameras on NASA's Mars Reconnaissance Orbiter will be used to gather more information about them before the final selection. The topographic map uses data from the Mars Orbiter Laser Altimeter on NASA's Mars Global Surveyor spacecraft. The color coding on this map indicates elevation relative to a reference datum, since Mars has no "sea level." The lowest elevations are presented as dark blue; the highest as white. The difference between green and orange in the color coding is about 2.5 miles (4 kilometers) vertically. Note: After thorough examination, NASA managers have decided to suspend the planned March 2016 launch of the Interior Exploration using

Heterogeneous Electrocatalyst with Molecular Cobalt Ions Serving as the Center of Active Sites.

PubMed

Wang, Jiong; Ge, Xiaoming; Liu, Zhaolin; Thia, Larissa; Yan, Ya; Xiao, Wei; Wang, Xin

2017-02-08

Molecular Co 2+ ions were grafted onto doped graphene in a coordination environment, resulting in the formation of molecularly well-defined, highly active electrocatalytic sites at a heterogeneous interface for the oxygen evolution reaction (OER). The S dopants of graphene are suggested to be one of the binding sites and to be responsible for improving the intrinsic activity of the Co sites. The turnover frequency of such Co sites is greater than that of many Co-based nanostructures and IrO 2 catalysts. Through a series of carefully designed experiments, the pathway for the evolution of the Co cation-based molecular catalyst for the OER was further demonstrated on such a single Co-ion site for the first time. The Co 2+ ions were successively oxidized to Co 3+ and Co 4+ states prior to the OER. The sequential oxidation was coupled with the transfer of different numbers of protons/hydroxides and generated an active Co 4+ ═O fragment. A side-on hydroperoxo ligand of the Co 4+ site is proposed as a key intermediate for the formation of dioxygen.

Digestive enzyme activities in the guts of bonnethead sharks (Sphyrna tiburo) provide insight into their digestive strategy and evidence for microbial digestion in their hindguts.

PubMed

Jhaveri, Parth; Papastamatiou, Yannis P; German, Donovan P

2015-11-01

Few investigations have studied digestive enzyme activities in the alimentary tracts of sharks to gain insight into how these organisms digest their meals. In this study, we examined the activity levels of proteases, carbohydrases, and lipase in the pancreas, and along the anterior intestine, spiral intestine, and colon of the bonnethead shark, Sphyrna tiburo. We then interpreted our data in the context of a rate-yield continuum to discern this shark's digestive strategy. Our data show anticipated decreasing patterns in the activities of pancreatic enzymes moving posteriorly along the gut, but also show mid spiral intestine peaks in aminopeptidase and lipase activities, which support the spiral intestine as the main site of absorption in bonnetheads. Interestingly, we observed spikes in the activity levels of N-acetyl-β-D-glucosaminidase and β-glucosidase in the bonnethead colon, and these chitin- and cellulose-degrading enzymes, respectively, are likely of microbial origin in this distal gut region. Taken in the context of intake and relatively long transit times of food through the gut, the colonic spikes in N-acetyl-β-D-glucosaminidase and β-glucosidase activities suggest that bonnetheads take a yield-maximizing strategy to the digestive process, with some reliance on microbial digestion in their hindguts. This is one of the first studies to examine digestive enzyme activities along the gut of any shark, and importantly, the data match with previous observations that sharks take an extended time to digest their meals (consistent with a yield-maximizing digestive strategy) and that the spiral intestine is the primary site of absorption in sharks. Copyright © 2015 Elsevier Inc. All rights reserved.

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes*

PubMed Central

Lopata, Anna; Leveles, Ibolya; Bendes, Ábris Ádám; Viskolcz, Béla; Vértessy, Beáta G.; Jójárt, Balázs; Tóth, Judit

2016-01-01

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent. PMID:27815500

Pyrrole-Based Antitubulin Agents: Two Distinct Binding Modalities Are Predicted for C-2 Analogues in the Colchicine Site

PubMed Central

2011-01-01

3,5-Dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogues were synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct binding modes that explain the structure–activity relationships and are in accord with the structural basis of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the colchicine site and compound design. PMID:22611477

Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA

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

Sidhu, Navdeep S.; University of Göttingen, Tammannstrasse 4, 37077 Göttingen; Schreiber, Kathrin

2014-05-01

Mucopolysaccharidosis IIIA is a fatal neurodegenerative disease that typically manifests itself in childhood and is caused by mutations in the gene for the lysosomal enzyme sulfamidase. The first structure of this enzyme is presented, which provides insight into the molecular basis of disease-causing mutations, and the enzymatic mechanism is proposed. Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However,more » the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.« less

Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

PubMed

Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

2016-03-01

Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

PubMed Central

Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

1992-01-01

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

HIV integration sites and implications for maintenance of the reservoir.

PubMed

Symons, Jori; Cameron, Paul U; Lewin, Sharon R

2018-03-01

To provide an overview of recent research of how HIV integration relates to productive and latent infection and implications for cure strategies. How and where HIV integrates provides new insights into how HIV persists on antiretroviral therapy (ART). Clonal expansion of infected cells with the same integration site demonstrates that T-cell proliferation is an important factor in HIV persistence, however, the driver of proliferation remains unclear. Clones with identical integration sites harbouring defective provirus can accumulate in HIV-infected individuals on ART and defective proviruses can express RNA and produce protein. HIV integration sites differ in clonally expanded and nonexpanded cells and in latently and productively infected cells and this influences basal and inducible transcription. There is a growing number of cellular proteins that can alter the pattern of integration to favour latency. Understanding these pathways may identify new interventions to eliminate latently infected cells. Using advances in analysing HIV integration sites, T-cell proliferation of latently infected cells is thought to play a major role in HIV persistence. Clonal expansion has been demonstrated with both defective and intact viruses. Production of viral RNA and protein from defective viruses may play a role in driving chronic immune activation. The site of integration may determine the likelihood of proliferation and the degree of basal and induced transcription. Finally, host factors and gene expression at the time of infection may determine the integration site. Together these new insights may lead to novel approaches to elimination of latently infected cells.

Insight in Schizophrenia: Involvement of Self-Reflection Networks?

PubMed Central

de Vos, Annerieke E.

2013-01-01

Background: Impaired insight is a common feature in psychosis and an important predictor of variables such as functional outcome, prognosis, and treatment adherence. A cognitive process that may underlie insight in psychosis is self-reflection, or the conscious evaluation of one’s traits and characteristics. The current study aims to investigate the neural correlates of self-reflective processing and its relationship with insight in schizophrenia. Methods: Forty-seven schizophrenia patients and 21 healthy controls performed a self-reflection task in a functional magnetic resonance imaging (fMRI) scanner. The tasks comprised a self-reflection, close other-reflection, and a semantic (baseline) condition. Insight scores were obtained with the Schedule of Assessment of Insight Expanded. In addition, cognitive insight scores were obtained (Beck Cognitive Insight Scale [BCIS]). Results: Schizophrenia patients demonstrated less activation in the posterior cingulate cortex in the self- and other-reflection conditions and less activation in the precuneus in the other-reflection condition compared with healthy controls. Better insight was associated with greater response in the inferior frontal gyrus, anterior insula, and inferior parietal lobule during self-reflection. In addition, better cognitive insight was associated with higher activation in ventromedial prefrontal cortex during self-reflection. Conclusion: In the current study, evidence for a relationship between self-reflection and insight in patients with schizophrenia was found in brain areas related to self-reflection, self/other distinction and source attribution. The findings support the rationale for a treatment that is currently under evaluation, which attempts to increase insight by enhancing self-reflection. PMID:23104865

Human γ-Glutamyl Transpeptidase 1: STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS.

PubMed

Terzyan, Simon S; Burgett, Anthony W G; Heroux, Annie; Smith, Clyde A; Mooers, Blaine H M; Hanigan, Marie H

2015-07-10

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal.

PubMed

Jain, Rinku; Hao, Bing; Liu, Ren-Peng; Chan, Michael K

2005-04-06

E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.

Strategy for selecting Mars Pathfinder landing sites

NASA Technical Reports Server (NTRS)

Greeley, Ronald; Kuzmin, Ruslin O.

1994-01-01

A strategy for Pathfinder site selection must be developed that is fundamentally different from most previous considerations. At least two approaches can be identified. In one approach, the objective is to select a site representing a key geologic unit on Mars, i.e., a unit that is widespread, easily recognized, and used frequently as a datum in various investigations. The second approach is to select a site that potentially affords access to a wide variety of rock types. Because rover range is limited, rocks from a variety of sources must be assembled in a small area for sampling. Regardless of the approach taken in site selection, the Pathfinder site should include eolian deposits and provisions should be made to obtain measurements on soils. A recommended approach for selecting the Mars Pathfinder landing site is to identify a deltaic deposit, composed of sediments derived from sources of various ages and geologic units that shows evidence of eolian activity. The site should be located as close as possible to the part of the outwash where rapid deposition occurred because the likelihood of 'sorting' by size and composition increases with distance, decreasing the probability of heterogeneity. In addition, it is recommended that field operation tests be conducted to gain experience and insight into conducting science with Pathfinder.

A Comparative Analysis of Synthetic Quorum Sensing Modulators in Pseudomonas aeruginosa: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design.

PubMed

Moore, Joseph D; Rossi, Francis M; Welsh, Michael A; Nyffeler, Kayleigh E; Blackwell, Helen E

2015-11-25

Quorum sensing (QS) is a chemical signaling mechanism that allows bacterial populations to coordinate gene expression in response to social and environmental cues. Many bacterial pathogens use QS to initiate infection at high cell densities. Over the past two decades, chemical antagonists of QS in pathogenic bacteria have attracted substantial interest for use both as tools to further elucidate QS mechanisms and, with further development, potential anti-infective agents. Considerable recent research has been devoted to the design of small molecules capable of modulating the LasR QS receptor in the opportunistic pathogen Pseudomonas aeruginosa. These molecules hold significant promise in a range of contexts; however, as most compounds have been developed independently, comparative activity data for these compounds are scarce. Moreover, the mechanisms by which the bulk of these compounds act are largely unknown. This paucity of data has stalled the choice of an optimal chemical scaffold for further advancement. Herein, we submit the best-characterized LasR modulators to standardized cell-based reporter and QS phenotypic assays in P. aeruginosa, and we report the first comprehensive set of comparative LasR activity data for these compounds. Our experiments uncovered multiple interesting mechanistic phenomena (including a potential alternative QS-modulatory ligand binding site/partner) that provide new, and unexpected, insights into the modes by which many of these LasR ligands act. The lead compounds, data trends, and mechanistic insights reported here will significantly aid the design of new small molecule QS inhibitors and activators in P. aeruginosa, and in other bacteria, with enhanced potencies and defined modes of action.

Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

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

Wubben, T.; Mesecar, A.D.; UIC)

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observedmore » in the MtPPAT-CoA complex.« less

Spontaneous neural activity in the right superior temporal gyrus and left middle temporal gyrus is associated with insight level in obsessive-compulsive disorder.

PubMed

Fan, Jie; Zhong, Mingtian; Gan, Jun; Liu, Wanting; Niu, Chaoyang; Liao, Haiyan; Zhang, Hongchun; Tan, Changlian; Yi, Jinyao; Zhu, Xiongzhao

2017-01-01

Insight into illness is an important issue for psychiatry disorder. Although the existence of a poor insight subtype of obsessive-compulsive disorder (OCD) was recognized in the DSM-IV, and the insight level in OCD was specified further in DSM-V, the neural underpinnings of insight in OCD have been rarely explored. The present study was designed to bridge this research gap by using resting-state functional magnetic resonance imaging (fMRI). Spontaneous neural activity were examined in 19 OCD patients with good insight (OCD-GI), 18 OCD patients with poor insight (OCD-PI), and 25 healthy controls (HC) by analyzing the amplitude of low-frequency fluctuation (ALFF) in the resting state. Pearson correlation analysis was performed between regional ALFFs and insight levels among OCD patients. OCD-GI and OCD-PI demonstrated overlapping and distinct brain alterations. Notably, compared with OCD-GI, tOCD-PI had reduced ALFF in left middle temporal gyrus (MTG) and right superior temporal gyrus (STG), as well as increased ALFF in right middle occipital gyrus. Further analysis revealed that ALFF values for the left MTG and right STG were correlated negatively with insight level in patients with OCD. Relatively small sample size and not all patients were un-medicated are our major limitations. Spontaneous brain activity in left MTG and right STG may be neural underpinnings of insight in OCD. Our results suggest the great role of human temporal brain regions in understanding insight, and further underscore the importance of considering insight presentation in understanding the clinical heterogeneity of OCD. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

PubMed

Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

2012-04-20

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

Synthesis of a molecularly defined single-active site heterogeneous catalyst for selective oxidation of N-heterocycles.

PubMed

Zhang, Yujing; Pang, Shaofeng; Wei, Zhihong; Jiao, Haijun; Dai, Xingchao; Wang, Hongli; Shi, Feng

2018-04-13

Generally, a homogeneous catalyst exhibits good activity and defined active sites but it is difficult to recycle. Meanwhile, a heterogeneous catalyst can easily be reused but its active site is difficult to reveal. It is interesting to bridge the gap between homogeneous and heterogeneous catalysis via controllable construction of a heterogeneous catalyst containing defined active sites. Here, we report that a molecularly defined, single-active site heterogeneous catalyst has been designed and prepared via the oxidative polymerization of maleimide derivatives. These polymaleimide derivatives can be active catalysts for the selective oxidation of heterocyclic compounds to quinoline and indole via the recycling of -C=O and -C-OH groups, which was confirmed by tracing the reaction with GC-MS using maleimide as the catalyst and by FT-IR analysis with polymaleimide as the catalyst. These results might promote the development of heterogeneous catalysts with molecularly defined single active sites exhibiting a comparable activity to homogeneous catalysts.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

PubMed

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Calcium-Activated Cl- Channel: Insights on the Molecular Identity in Epithelial Tissues.

PubMed

Rottgen, Trey S; Nickerson, Andrew J; Rajendran, Vazhaikkurichi M

2018-05-10

Calcium-activated chloride secretion in epithelial tissues has been described for many years. However, the molecular identity of the channel responsible for the Ca 2+ -activated Cl − secretion in epithelial tissues has remained a mystery. More recently, TMEM16A has been identified as a new putative Ca 2+ -activated Cl − channel (CaCC). The primary goal of this article will be to review the characterization of TMEM16A, as it relates to the physical structure of the channel, as well as important residues that confer voltage and Ca 2+ -sensitivity of the channel. This review will also discuss the role of TMEM16A in epithelial physiology and potential associated-pathophysiology. This will include discussion of developed knockout models that have provided much needed insight on the functional localization of TMEM16A in several epithelial tissues. Finally, this review will examine the implications of the identification of TMEM16A as it pertains to potential novel therapies in several pathologies.

Trace element geochemistry in archaeological sites.

PubMed

Jenkins, D A

1989-06-01

Man can leave a geochemical imprint on an archaeological site in several ways. In common with other components of the biosphere, there is a selective enrichment of elements in his body tissues which, upon death and burial, may lead to detectable anomalies. Of elements concentrated in this way P is the most obvious, but Sn could be another possibility worth further investigation. There has been particular geochemical impact due to the progressive use of such metals as Ag, Au, Cu, Pb and Zn during successive cultural stages. Anomalies may thus arise due to recognisable transported ore, slags or artefacts, although there has also often been cryptic redispersion of the metals within a site. Charcoal is one of the commoner finds during excavations, and it has the ability to adsorb and concentrate metals progressively from percolating solutions since the time of its burial: with careful interpretation its analysis may thus provide a valuable historical record, as is illustrated by material from several sites in North Wales. Providing care is taken to interpret results in their particular geochemical and pedochemical context, trace element analysis may thus offer a useful insight into the history of man's activities in an archaeological site.

Interactive flare sites within an active region complex

NASA Technical Reports Server (NTRS)

Poletto, G.; Gary, G. A.; Machado, M. E.

1993-01-01

We examine here a set of images of an active region complex, acquired on June 24-25, 1980, by the Hard X-ray Imaging Spectrometer on SMM, with the purpose of establishing whether there was any interplay between the frequent activity observed at different sites in the activity center and, in such a case, how the interaction was established. By analyzing both quiet and active orbits we show that, as a rule, activity originating in one region triggers the other region's activity. However, we find little unambiguous evidence for the presence of large-scale interconnecting loops. A comparison of X-ray images with magnetic field observations suggested that we interpret the active region behavior in terms of the interaction between different loop systems, in a scenario quite analogous to the interacting bipole representation of individual flares. We conclude that active region interplay provides an easily observable case to study the time-dependent topology and the mechanisms for the spreading of activity in transient events over all energy scales.

New insights into insight: Neurophysiological correlates of the difference between the intrinsic "aha" and the extrinsic "oh yes" moment.

PubMed

Rothmaler, Katrin; Nigbur, Roland; Ivanova, Galina

2017-01-27

Insight refers to a situation in which a problem solver immediately changes his understanding of a problem situation. This representational change can either be triggered by external stimuli, like a hint or the solution itself, or by internal solution attempts. In the present paper, the differences and similarities between these two phenomena, namely "extrinsic" and "intrinsic" insight, are examined. To this end, electroencephalogram (EEG) is recorded while subjects either recognize or generate solutions to German verbal compound remote associate problems (CRA). Based on previous studies, we compare the alpha power prior to insightful solution recognition with the alpha power prior to insightful solution generation. Results show that intrinsic insights are preceded by an increase in alpha power at right parietal electrodes, while extrinsic insights are preceded by a respective decrease. These results can be interpreted in two ways. In consistency with other studies, the increase in alpha power before intrinsic insights can be interpreted as an increased internal focus of attention. Accordingly, the decrease in alpha power before extrinsic insights may be associated with a more externally oriented focus of attention. Alternatively, the increase in alpha power prior to intrinsic insights can be interpreted as an active inhibition of solution-related information, while the alpha power decrease prior to extrinsic insights may reflect its activation. Regardless of the interpretation, the results provide strong evidence that extrinsic and intrinsic insight differ on the behavioral as well as the neurophysiological level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

Is creative insight task-specific? A coordinate-based meta-analysis of neuroimaging studies on insightful problem solving.

PubMed

Shen, Wangbing; Yuan, Yuan; Liu, Chang; Zhang, Xiaojiang; Luo, Jing; Gong, Zhe

2016-12-01

The question of whether creative insight varies across problem types has recently come to the forefront of studies of creative cognition. In the present study, to address the nature of creative insight, the coordinate-based activation likelihood estimation (ALE) technique was utilized to individually conduct three quantitative meta-analyses of neuroimaging experiments that used the compound remote associate (CRA) task, the prototype heuristic (PH) task and the Chinese character chunk decomposition (CCD) task. These tasks were chosen because they are frequently used to uncover the neurocognitive correlates of insight. Our results demonstrated that creative insight reliably activates largely non-overlapping brain regions across task types, with the exception of some shared regions: the CRA task mainly relied on the right parahippocampal gyrus, the superior frontal gyrus and the inferior frontal gyrus; the PH task primarily depended on the right middle occipital gyrus (MOG), the bilateral superior parietal lobule/precuneus, the left inferior parietal lobule, the left lingual gyrus and the left middle frontal gyrus; and the CCD task activated a broad cerebral network consisting of most dorsolateral and medial prefrontal regions, frontoparietal regions and the right MOG. These results provide the first neural evidence of the task dependence of creative insight. The implications of these findings for resolving conflict surrounding the different theories of creative cognition and for defining insight as a set of heterogeneous processes are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering streptokinase for generation of active site-labeled plasminogen analogs*

PubMed Central

Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

2011-01-01

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

PubMed Central

Das, Aditi; Mandal, Chhabinath; Dasgupta, Arindam; Sengupta, Tanushri; Majumder, Hemanta K.

2002-01-01

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes. PMID:11809893

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor*

PubMed Central

Daly, Norelle L.; Thorstholm, Louise; Greenwood, Kathryn P.; King, Gordon J.; Rosengren, K. Johan; Heras, Begoña; Martin, Jennifer L.; Craik, David J.

2013-01-01

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin. PMID:24169696

HPLC-Based Activity Profiling: Discovery of Piperine as a Positive GABAA Receptor Modulator Targeting a Benzodiazepine-Independent Binding Site

PubMed Central

Zaugg, Janine; Baburin, Igor; Strommer, Barbara; Kim, Hyun-Jung; Hering, Steffen; Hamburger, Matthias

2011-01-01

A plant extract library was screened for GABAA receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 μg/mL] potentiated GABA-induced chloride currents through GABAA receptors (composed of α1, β2, and γ2S subunits) by 169.1 ± 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1–4, 6–13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 ± 26.5% with an EC50 of 52.4 ± 9.4 μM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABAA receptor modulation. The stimulation of chloride currents through GABAA receptors by compound 5 was not antagonized by flumazenil (10 μM). These data show that piperine (5) represents a new scaffold of positive allosteric GABAA receptor modulators targeting a benzodiazepine-independent binding site. PMID:20085307

Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

PubMed Central

Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

2015-01-01

Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

PubMed Central

tenBrink, Felix; Schink, Bernhard; Kroneck, Peter M. H.

2011-01-01

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme. PMID:21193613

Baseline and premining geochemical characterization of mined sites

USGS Publications Warehouse

Nordstrom, D. Kirk

2015-01-01

A rational goal for environmental restoration of new, active, or inactive mine sites would be ‘natural background’ or the environmental conditions that existed before any mining activities or other related anthropogenic activities. In a strictly technical sense, there is no such thing as natural background (or entirely non-anthropogenic) existing today because there is no part of the planet earth that has not had at least some chemical disturbance from anthropogenic activities. Hence, the terms ‘baseline’ and ‘pre-mining’ are preferred to describe these conditions. Baseline conditions are those that existed at the time of the characterization which could be pre-mining, during mining, or post-mining. Protocols for geochemically characterizing pre-mining conditions are not well-documented for sites already mined but there are two approaches that seem most direct and least ambiguous. One is characterization of analog sites along with judicious application of geochemical modeling. The other is reactive-transport modeling (based on careful synoptic sampling with tracer-injection) and subtracting inputs from known mining and mineral processing. Several examples of acidic drainage are described from around the world documenting the range of water compositions produced from pyrite oxidation in the absence of mining. These analog sites provide insight to the processes forming mineralized waters in areas untouched by mining. Natural analog water-chemistry data is compared with the higher metal concentrations, metal fluxes, and weathering rates found in mined areas in the few places where comparisons are possible. The differences are generally 1–3 orders of magnitude higher for acid mine drainage.

Comparison of the active-site design of molybdenum oxo-transfer enzymes by quantum mechanical calculations.

PubMed

Li, Jilai; Ryde, Ulf

2014-11-17

There are three families of mononuclear molybdenum enzymes that catalyze oxygen atom transfer (OAT) reactions, named after a typical example from each family, viz., dimethyl sulfoxide reductase (DMSOR), sulfite oxidase (SO), and xanthine oxidase (XO). These families differ in the construction of their active sites, with two molybdopterin groups in the DMSOR family, two oxy groups in the SO family, and a sulfido group in the XO family. We have employed density functional theory calculations on cluster models of the active sites to understand the selection of molybdenum ligands in the three enzyme families. Our calculations show that the DMSOR active site has a much stronger oxidative power than the other two sites, owing to the extra molybdopterin ligand. However, the active sites do not seem to have been constructed to make the OAT reaction as exergonic as possible, but instead to keep the reaction free energy close to zero (to avoid excessive loss of energy), thereby making the reoxidation (SO and XO) or rereduction of the active sites (DMSOR) after the OAT reaction facile. We also show that active-site models of the three enzyme families can all catalyze the reduction of DMSO and that the DMSOR model does not give the lowest activation barrier. Likewise, all three models can catalyze the oxidation of sulfite, provided that the Coulombic repulsion between the substrate and the enzyme model can be overcome, but for this harder reaction, the SO model gives the lowest activation barrier, although the differences are not large. However, only the XO model can catalyze the oxidation of xanthine, owing to its sulfido ligand.

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

PubMed

Bate, Paul; Warwicker, Jim

2004-07-02

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

PubMed

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

The contribution of extracurricular activities to adolescent friendships: new insights through social network analysis.

PubMed

Schaefer, David R; Simpkins, Sandra D; Vest, Andrea E; Price, Chara D

2011-07-01

Extracurricular activities are settings that are theorized to help adolescents maintain existing friendships and develop new friendships. The overarching goal of the current investigation was to examine whether coparticipating in school-based extracurricular activities supported adolescents' school-based friendships. We used social network methods and data from the National Longitudinal Study of Adolescent Health to examine whether dyadic friendship ties were more likely to exist among activity coparticipants while controlling for alternative friendship processes, namely dyadic homophily (e.g., demographic and behavioral similarities) and network-level processes (e.g., triadic closure). Results provide strong evidence that activities were associated with current friendships and promoted the formation of new friendships. These associations varied based on school level (i.e., middle vs. high school) and activity type (i.e., sports, academic, arts). Results of this study provide new insight into the complex relations between activities and friendship that can inform theories of their developmental outcomes. PsycINFO Database Record (c) 2011 APA, all rights reserved

The Contribution of Extracurricular Activities to Adolescent Friendships: New Insights through Social Network Analysis

PubMed Central

Schaefer, David R.; Simpkins, Sandra D.; Vest, Andrea E.; Price, Chara D.

2011-01-01

Extracurricular activities are settings that are theorized to help adolescents maintain existing friendships and develop new friendships. The overarching goal of the current investigation was to examine whether co-participating in school-based extracurricular activities supported adolescents’ school-based friendships. We utilized social network methods and data from the National Longitudinal Study of Adolescent Health to examine whether dyadic friendship ties were more likely to exist among activity co-participants while controlling for alternative friendship processes, namely dyadic homophily (e.g., demographic and behavioral similarities) and network-level processes (e.g., triadic closure). Results provide strong evidence that activities were associated with current friendships and promoted the formation of new friendships. These associations varied based on school level (i.e., middle versus high school) and activity type (i.e., sports, academic, arts). Results of this study provide new insight into the complex relations between activities and friendship that can inform theories of their developmental outcomes. PMID:21639618

Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

ERIC Educational Resources Information Center

Heo, Gyeong Mi; Lee, Romee

2013-01-01

This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

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

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Identification and characterization of the sodium-binding site of activated protein C.

PubMed

He, X; Rezaie, A R

1999-02-19

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

Patched bimetallic surfaces are active catalysts for ammonia decomposition

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

Guo, Wei; Vlachos, Dionisios G.

In this study, ammonia decomposition is often used as an archetypical reaction for predicting new catalytic materials and understanding the very reason of why some reactions are sensitive on material’s structure. Core–shell or surface-segregated bimetallic nanoparticles expose outstanding activity for many heterogeneously catalysed reactions but the reasons remain elusive owing to the difficulties in experimentally characterizing active sites. Here by performing multiscale simulations in ammonia decomposition on various nickel loadings on platinum (111), we show that the very high activity of core–shell structures requires patches of the guest metal to create and sustain dual active sites: nickel terraces catalyse N-Hmore » bond breaking and nickel edge sites drive atomic nitrogen association. The structure sensitivity on these active catalysts depends profoundly on reaction conditions due to kinetically competing relevant elementary reaction steps. We expose a remarkable difference in active sites between transient and steady-state studies and provide insights into optimal material design.« less

Patched bimetallic surfaces are active catalysts for ammonia decomposition

DOE PAGES

Guo, Wei; Vlachos, Dionisios G.

2015-10-07

In this study, ammonia decomposition is often used as an archetypical reaction for predicting new catalytic materials and understanding the very reason of why some reactions are sensitive on material’s structure. Core–shell or surface-segregated bimetallic nanoparticles expose outstanding activity for many heterogeneously catalysed reactions but the reasons remain elusive owing to the difficulties in experimentally characterizing active sites. Here by performing multiscale simulations in ammonia decomposition on various nickel loadings on platinum (111), we show that the very high activity of core–shell structures requires patches of the guest metal to create and sustain dual active sites: nickel terraces catalyse N-Hmore » bond breaking and nickel edge sites drive atomic nitrogen association. The structure sensitivity on these active catalysts depends profoundly on reaction conditions due to kinetically competing relevant elementary reaction steps. We expose a remarkable difference in active sites between transient and steady-state studies and provide insights into optimal material design.« less

Patched bimetallic surfaces are active catalysts for ammonia decomposition.

PubMed

Guo, Wei; Vlachos, Dionisios G

2015-10-07

Ammonia decomposition is often used as an archetypical reaction for predicting new catalytic materials and understanding the very reason of why some reactions are sensitive on material's structure. Core-shell or surface-segregated bimetallic nanoparticles expose outstanding activity for many heterogeneously catalysed reactions but the reasons remain elusive owing to the difficulties in experimentally characterizing active sites. Here by performing multiscale simulations in ammonia decomposition on various nickel loadings on platinum (111), we show that the very high activity of core-shell structures requires patches of the guest metal to create and sustain dual active sites: nickel terraces catalyse N-H bond breaking and nickel edge sites drive atomic nitrogen association. The structure sensitivity on these active catalysts depends profoundly on reaction conditions due to kinetically competing relevant elementary reaction steps. We expose a remarkable difference in active sites between transient and steady-state studies and provide insights into optimal material design.

Patched bimetallic surfaces are active catalysts for ammonia decomposition

NASA Astrophysics Data System (ADS)

Guo, Wei; Vlachos, Dionisios G.

2015-10-01

Ammonia decomposition is often used as an archetypical reaction for predicting new catalytic materials and understanding the very reason of why some reactions are sensitive on material's structure. Core-shell or surface-segregated bimetallic nanoparticles expose outstanding activity for many heterogeneously catalysed reactions but the reasons remain elusive owing to the difficulties in experimentally characterizing active sites. Here by performing multiscale simulations in ammonia decomposition on various nickel loadings on platinum (111), we show that the very high activity of core-shell structures requires patches of the guest metal to create and sustain dual active sites: nickel terraces catalyse N-H bond breaking and nickel edge sites drive atomic nitrogen association. The structure sensitivity on these active catalysts depends profoundly on reaction conditions due to kinetically competing relevant elementary reaction steps. We expose a remarkable difference in active sites between transient and steady-state studies and provide insights into optimal material design.

Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

PubMed Central

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

2011-01-01

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

DOE Data Explorer

John Peterson

2015-04-17

These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

PubMed Central

Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

2015-01-01

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

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

Tomkinson, B.; Wernstedt, C.; Hellman, U.

1987-11-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residuesmore » 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.« less

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

PubMed Central

Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

1987-01-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

PubMed Central

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

PubMed

Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

2013-05-10

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

PubMed

Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

PubMed Central

Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

PubMed

Sen, Kakali; Horrell, Sam; Kekilli, Demet; Yong, Chin W; Keal, Thomas W; Atakisi, Hakan; Moreau, David W; Thorne, Robert E; Hough, Michael A; Strange, Richard W

2017-07-01

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

DOE PAGES

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open'more » structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. In conclusion, as a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

PubMed

Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

PubMed

Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

2018-06-01

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

PubMed

Fenwick, Michael K; Mehta, Angad P; Zhang, Yang; Abdelwahed, Sameh H; Begley, Tadhg P; Ealick, Steven E

2015-03-27

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

Clinical correlates of loss of insight in bipolar depression.

PubMed

Silva, Rafael de Assis da; Mograbi, Daniel C; Camelo, Evelyn V M; Santana, Cristina M T; Landeira-Fernandez, Jesus; Cheniaux, Elie

2017-01-01

Affective state may influence insight, especially regarding mania. Nevertheless, studies have so far suggested that depression seems not to significantly impair insight. To the best of our knowledge, this study pioneers the evaluation of how insight variations in bipolar depression correlate with clinical variables. A group of 165 bipolar patients, 52 of whom had depressive episodes according to DSM-5 criteria, were followed during a year. All patients underwent clinical assessment, and insight was evaluated through the Insight Scale for Affective Disorders (ISAD). Repeated-measures ANOVA was calculated comparing scores on the four ISAD factors (insight into symptoms, the condition itself, self-esteem and social relationships) in order to investigate differences in insight according to different objects. Correlational analysis explored which clinical symptoms were linked to reduced insight. Worse total insight correlated with suicide attempt/ideation and fewer subsyndromal manic symptoms such as mood elevation, increased energy and sexual interest. Worse self-esteem insight was associated with not only suicide ideation/attempt but also with activity reduction and psychomotor retardation. Worse symptom insight also correlated with psychomotor retardation. Better insight into having an affective disorder was associated with more intense hypochondria symptoms. Finally, worse insight into having an illness was associated with psychotic episodes. Our study found that symptoms other than psychosis - suicide ideation, psychomotor retardation and reduction of activity and work - correlate with insight impairment in bipolar depression.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

PubMed

Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

2016-10-04

Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

Well-Defined Metal-O6 in Metal-Catecholates as a Novel Active Site for Oxygen Electroreduction.

PubMed

Liu, Xuan-He; Hu, Wei-Li; Jiang, Wen-Jie; Yang, Ya-Wen; Niu, Shuai; Sun, Bing; Wu, Jing; Hu, Jin-Song

2017-08-30

Metal-nitrogen coordination sites, M-N x (M = Fe, Co, Ni, etc.), have shown great potential to replace platinum group materials as electrocatalysts for oxygen reduction reaction (ORR). However, the real active site in M-N x is still vague to date due to their complicated structure and composition. It is therefore highly desirable but challenging to develop ORR catalysts with novel and clear active sites, which could meet the needs of comprehensive understanding of structure-function relationships and explore new cost-effective and efficient ORR electrocatalysts. Herein, well-defined M-O 6 coordination in metal-catecholates (M-CATs, M = Ni or Co) is discovered to be catalytically active for ORR via a four-electron-dominated pathway. In view of no pyrolysis involved and unambiguous crystalline structure of M-CATs, the M-O 6 octahedral coordination site with distinct structure is determined as a new type of active site for ORR. These findings extend the scope of metal-nonmetal coordination as an active site for ORR and pave a way for bottom-up design of novel electrocatalysts containing M-O 6 coordination.

Insights into structure–activity relationship of GABAA receptor modulating coumarins and furanocoumarins

PubMed Central

Singhuber, Judith; Baburin, Igor; Ecker, Gerhard F.; Kopp, Brigitte; Hering, Steffen

2011-01-01

The coumarins imperatorin and osthole are known to exert anticonvulsant activity. We have therefore analyzed the modulation of GABA-induced chloride currents (IGABA) by a selection of 18 coumarin derivatives on recombinant α1β2γ2S GABAA receptors expressed in Xenopus laevis oocytes by means of the two-microelectrode voltage clamp technique. Osthole (EC50=14±1 μM) and oxypeucedanin (EC50=25±8 μM) displayed the highest efficiency with IGABA potentiation of 116±4% and 547±56%, respectively. IGABA enhancement by osthole and oxypeucedanin was not inhibited by flumazenil (1 μM) indicating an interaction with a binding site distinct from the benzodiazepine binding site. In general, prenyl residues are essential for the positive modulatory activity, while longer side chains or bulkier residues (e.g. geranyl residues) diminish IGABA modulation. Generation of a binary classification tree revealed the importance of polarisability, which is sufficient to distinguish actives from inactives. A 4-point pharmacophore model based on oxypeucedanin – comprising three hydrophobic and one aromatic feature – identified 6 out of 7 actives as hits. In summary, (oxy-)prenylated coumarin derivatives from natural origin represent new GABAA receptor modulators. PMID:21749864

Enhancement of catalytic activity and thermostability of a thermostable cellobiohydrolase from Chaetomium thermophilum by site-directed mutagenesis.

PubMed

Han, Chao; Li, Weiguang; Hua, Chengyao; Sun, Fengqing; Bi, Pengsheng; Wang, Qunqing

2018-05-20

Enzymatic saccharification of lignocellulosic biomass is increasingly applied in agricultural and industrial applications. Nevertheless, low performance in the extreme environment severely prevents the utilization of commercial enzyme preparations. To obtain cellobiohydrolases with improved catalytic activity and thermostability, structure-based rational design was performed based on a thermostable cellobiohydrolase CtCel6 from Chaetomium thermophilum. In the present study, four conserved and noncatalytic residue substitutions were generated via site-directed mutagenesis. Mutations were heterologously expressed in yeast Pichia pastoris, purified, and ultimately assayed for enzymatic characteristics. The mutant Y119F increased the catalytic activity 1.82-, 1.65- and 1.43-fold against β-d-glucan, phosphoric acid swollen cellulose (PASC) and carboxymethylcellulose sodium (CMC-Na), respectively. In addition, S131 W effectively enhanced the enzyme's heat resistance to elevated temperatures. The half-life (t 1/2 ) of this mutant enzyme was increased 1.42- and 2.40-fold at 80 °C and 90 °C, respectively, compared to the wild-type. This study offers initial insight into the biological function of the conserved and noncatalytic residues of thermostable cellobiohydrolases and provides a valid approach to the improvement of enzyme redesign proposal. Copyright © 2018 Elsevier B.V. All rights reserved.

Active chemisorption sites in functionalized ionic liquids for carbon capture.

PubMed

Cui, Guokai; Wang, Jianji; Zhang, Suojiang

2016-07-25

Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

PubMed

Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

2011-07-01

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP–aluminum fluoride: insights into activator mechanochemical action

PubMed Central

Chaney, Matthew; Grande, Ricardo; Wigneshweraraj, Siva R.; Cannon, Wendy; Casaz, Paul; Gallegos, Maria-Trinidad; Schumacher, Jorg; Jones, Susan; Elderkin, Sarah; Dago, Angel Ernesto; Morett, Enrique; Buck, Martin

2001-01-01

Conformational changes in sigma 54 (ς54) and ς54-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that ς54 and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP–aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of ς54 Region I to activator in the presence of ADP–aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to ς54 Region I. ADP–aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action. PMID:11544185

Probing the Active Surface Sites for CO Reduction on Oxide-Derived Copper Electrocatalysts

DOE PAGES

Verdaguer-Casadevall, Arnau; Li, Christina W.; Johansson, Tobias P.; ...

2015-07-30

CO electroreduction activity on oxide-derived Cu (OD-Cu) was found to correlate with metastable surface features that bind CO strongly. OD-Cu electrodes prepared by H 2 reduction of Cu 2O precursors reduce CO to acetate and ethanol with nearly 50% Faradaic efficiency at moderate overpotential. Temperature-programmed desorption of CO on OD-Cu revealed the presence of surface sites with strong CO binding that are distinct from the terraces and stepped sites found on polycrystalline Cu foil. After annealing at 350 °C, the surface-area corrected current density for CO reduction is 44-fold lower and the Faradaic efficiency is less than 5%. These changesmore » are accompanied by a reduction in the proportion of strong CO binding sites. Here, we propose that the active sites for CO reduction on OD-Cu surfaces are strong CO binding sites that are supported by grain boundaries. Uncovering these sites is a first step toward understanding the surface chemistry necessary for efficient CO electroreduction.« less

Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors.

PubMed

Mateos-Diaz, E; Amara, S; Roussel, A; Longhi, S; Cambillau, C; Carrière, F

2017-01-01

Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases. © 2017 Elsevier Inc. All rights reserved.

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase*

PubMed Central

Chen, Yaozong; Sun, Yueru; Song, Haigang; Guo, Zhihong

2015-01-01

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes. PMID:26276389

Localization of sclerotic-type chronic graft-vs-host disease to sites of skin injury: potential insight into the mechanism of isomorphic and isotopic responses.

PubMed

Martires, Kathryn J; Baird, Kristin; Citrin, Deborah E; Hakim, Fran T; Pavletic, Steven Z; Cowen, Edward W

2011-09-01

The mechanisms responsible for the variable manifestations of chronic cutaneous graft-vs-host disease (cGVHD) are poorly understood. Localization of sclerotic-type chronic graft-vs-host disease to sites of skin injury (isomorphic and isotopic responses), a recognized phenomenon in morphea, suggests a potential common pathway between cGVHD and other sclerotic skin conditions. Four cases of sclerotic-type cGVHD developed at the site of disparate skin injuries (ionizing radiotherapy, repeated needle sticks, central catheter site, and varicella-zoster virus infection). We review the spectrum of previously reported cases of sclerotic and nonsclerotic cGVHD relating to external forces on the skin. Localization of sclerotic-type cGVHD may occur after many types of skin injury, including UV and ionizing radiotherapy, needle sticks, viral infection, and pressure or friction. Recognition of this phenomenon may be helpful for the early diagnosis of sclerotic disease. Recent insights into the immunological consequences of minor skin injury may provide important clues to the underlying pathogenesis of cGVHD-mediated skin disease.

Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

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

Phillips, J.; Warby, C; Whitby, F

2009-01-01

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connectedmore » by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.« less

A DFT Study of Tungsten-Methylidene Formation on a W/ZSM-5 Zeolite: The Metathesis Active Site.

PubMed

Maihom, Thana; Probst, Michael; Limtrakul, Jumras

2015-10-26

Tungsten-methylidene formation from ethene on either the W(IV) , W(V) , or W(VI) active sites of a W/ZSM-5 zeolite is investigated by using the M06-L functional. The reaction is assumed to proceed in two steps; the first step is the [2+2] cycloaddition between ethene and the W-O active site to form an oxametallacycle intermediate. The intermediate is then decomposed to produce the W-methylidene active site from the metathesis reaction. The overall activation barrier of the reaction on W(VI) (27.3 kcal mol(-1) ) is considerably lower than the ones for W(IV) and W(V) (69.4 and 37.1 kcal mol(-1) , respectively). Moreover, the reaction involving the W(VI) site also stabilizes intermediates and products to a larger extent than the ones on the W(IV) and W(V) sites. As a result, we have demonstrated that the reaction of the W-methylidene metathesis active site is both kinetically and thermodynamically favored to occur on the W(VI) active site of the zeolite. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The impact of active site mutations of South African HIV PR on drug resistance: Insight from molecular dynamics simulations, binding free energy and per-residue footprints.

PubMed

Ahmed, Shaimaa M; Maguire, Glenn E M; Kruger, Hendrik G; Govender, Thirumala

2014-04-01

Molecular dynamics simulations and binding free energy calculations were used to provide an understanding of the impact of active site drug-resistant mutations of the South African HIV protease subtype C (C-SA HIV PR), V82A and V82F/I84V on drug resistance. Unique per-residue interaction energy 'footprints' were developed to map the overall drug-binding profiles for the wild type and mutants. Results confirmed that these mutations altered the overall binding landscape of the amino acid residues not only in the active site region but also in the flaps as well. Four FDA-approved drugs were investigated in this study; these include ritonavir (RTV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV). Computational results compared against experimental findings were found to be complementary. Against the V82F/I84V variant, saquinavir, indinavir, and nelfinavir lose remarkable entropic contributions relative to both wild-type and V82A C-SA HIV PRs. The per-residue energy 'footprints' and the analysis of ligand-receptor interactions for the drug complexes with the wild type and mutants have also highlighted the nature of drug interactions. The data presented in this study will prove useful in the design of more potent inhibitors effective against drug-resistant HIV strains. © 2013 John Wiley & Sons A/S.

Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

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

Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.

Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

DOE PAGES

Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.; ...

2018-02-05

Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

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

Carra,J.; McHugh, C.; Mulligan, S.

2007-01-01

We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding tomore » a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.« less

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

PubMed

Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

2016-11-15

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-26

... DEPARTMENT OF ENERGY Reimbursement for Costs of Remedial Action at Active Uranium and Thorium...) acceptance of claims in FY 2011 from eligible active uranium and thorium processing site licensees for... incurred by licensees at active uranium and thorium processing sites to remediate byproduct material...

Activation of dioxygen by copper metalloproteins and insights from model complexes.

PubMed

Quist, David A; Diaz, Daniel E; Liu, Jeffrey J; Karlin, Kenneth D

2017-04-01

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing metalloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O 2 -activation in copper proteins are addressed.

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

PubMed Central

Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

2014-01-01

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

DOE PAGES

Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; ...

2015-03-25

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. Here, we mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation ofmore » altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. In conclusion, the overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.« less

Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

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

Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. Here, we mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation ofmore » altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. In conclusion, the overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.« less

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

PubMed

Krajewska, Barbara; Zaborska, Wiesława

2007-10-01

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme : the active site H103W mutation restores ADP-ribosyl cyclase activity†

PubMed Central

Kuhn, Isabelle; Kellenberger, Esther; Rognan, Didier; Lund, Frances E.; Muller-Steffner, Hélène; Schuber, Francis

2008-01-01

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes which are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products, i.e., a value larger than observed for other members of this family such as CD38. Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provide new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge however in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR. PMID:17002287

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

PubMed Central

Gorres, Kelly L.; Pua, Khian Hong; Raines, Ronald T.

2009-01-01

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change. PMID:19890397

Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

PubMed

Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

2015-03-01

Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

Transfer hydrogenation over sodium-modified ceria: Enrichment of redox sites active for alcohol dehydrogenation

DOE PAGES

Nelson, Nicholas C.; Boote, Brett W.; Naik, Pranjali; ...

2017-01-17

Ceria (CeO 2) and sodium-modified ceria (Ce-Na) were prepared through combustion synthesis. Palladium was deposited onto the supports (Pd/CeO 2 and Pd/Ce-Na) and their activity for the aqueous-phase transfer hydrogenation of phenol using 2-propanol under liquid flow conditions was studied. Pd/Ce-Na showed a marked increase (6×) in transfer hydrogenation activity over Pd/CeO 2. Material characterization indicated that water-stable sodium species were not doped into the ceria lattice, but rather existed as subsurface carbonates. Modification of ceria by sodium provided more adsorption and redox active sites (i.e. defects) for 2-propanol dehydrogenation. This effect was an intrinsic property of the Ce-Na supportmore » and independent of Pd. The redox sites active for 2-propanol dehydrogenation were thermodynamically equivalent on both supports/catalysts. At high phenol concentrations, the reaction was limited by 2-propanol adsorption. Furthermore, the difference in catalytic activity was attributed to the different numbers of 2-propanol adsorption and redox active sites on each catalyst.« less

Improved ethanol electrooxidation performance by shortening Pd-Ni active site distance in Pd-Ni-P nanocatalysts

NASA Astrophysics Data System (ADS)

Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

2017-01-01

Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (~5 nm) Pd-Ni-P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni-P heterodimers into Pd-Ni-P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.

Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

2015-04-01

On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

PubMed Central

Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

2011-01-01

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

PubMed

Jacewicz, Agata; Trzemecka, Anna; Guja, Kip E; Plochocka, Danuta; Yakubovskaya, Elena; Bebenek, Anna; Garcia-Diaz, Miguel

2013-01-01

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

PubMed

Ibrahim, Firas; Andre, Claire; Iutzeler, Anne; Guillaume, Yves Claude

2013-10-01

A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k(cat)) was increased while the Michaelis constant (K(m)) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.

Novel insights into freshwater hydrocarbon-rich sediments using metatranscriptomics: Opening the black box.

PubMed

Reid, Thomas; Chaganti, Subba Rao; Droppo, Ian G; Weisener, Christopher G

2018-06-01

Baseline biogeochemical surveys of natural environments is an often overlooked field of environmental studies. Too often research begins once contamination has occurred, with a knowledge gap as to how the affected area behaved prior to outside (often anthropogenic) influences. These baseline characterizations can provide insight into proposed bioremediation strategies crucial in cleaning up chemical spill sites or heavily mined regions. Hence, this study was conducted to survey the in-situ microbial activity within freshwater hydrocarbon-rich environments cutting through the McMurray formation - the geologic strata constituting the oil sands. We are the first to report in-situ functional variations among these freshwater microbial ecosystems using metatranscriptomics, providing insight into the in-situ gene expression within these naturally hydrocarbon-rich sites. Key genes involved in energy metabolism (nitrogen, sulfur and methane) and hydrocarbon degradation, including transcripts relating to the observed expression of methane oxidation are reported. This information provides better linkages between hydrocarbon impacted environments, closing knowledge gaps for optimizing not only oil sands mine reclamation but also enhancing microbial reclamation strategies in various freshwater environments. These finding can also be applied to existing contaminated environments, in need of efficient reclamation efforts. Copyright © 2018 Elsevier Ltd. All rights reserved.

Work-site wellness programmes in Sweden: a cross-sectional study of physical activity, self-efficacy, and health.

PubMed

Gånedahl, H; Zsaludek Viklund, P; Carlén, K; Kylberg, E; Ekberg, J

2015-05-01

In Sweden, a work-site wellness programme implies reimbursing some of the expenses for health-promoting activities. Although work-site wellness programmes are readily available in Sweden, a large number of employees elect not to participate. The aim of this study was to investigate the association of physical activity, self-reported general health assessment and self-efficacy with participation in a work-site wellness programme. A cross-sectional study design was used. An online questionnaire was distributed to employees of a manufacturing company with 2500 employees in southwest Sweden. Those who took advantage of the work-site wellness programme assessed their general health as better and had higher assessment of physical activity. The study showed that being enlisted also implies a higher level of physical activity and general health; however, the effect sizes of these correlations were small. Self-efficacy, i.e. perceived behavioural control, was not associated with participation in the work-site wellness programme. However, self-efficacy was correlated with both general health assessment and physical activity. A regression analysis to determine explanatory contributions to the general health assessment score showed no significant contribution from participation in a work-site wellness programme, but was instead explained by perceived behavioural control and physical activity. Given the small effect size of the difference in physical activity between participators and non-participators in the work-site wellness programme, it is probable that only a small proportion of participators changed their health-promoting activities as a result of the work-site wellness programme. Copyright © 2015 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

DOE PAGES

Holby, Edward F.; Taylor, Christopher D.

2015-03-19

We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH boundmore » structures have the highest calculated activity to date.« less

The Diverse AAA+ Machines that Repair Inhibited Rubisco Active Sites

PubMed Central

Mueller-Cajar, Oliver

2017-01-01

Gaseous carbon dioxide enters the biosphere almost exclusively via the active site of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This highly conserved catalyst has an almost universal propensity to non-productively interact with its substrate ribulose 1,5-bisphosphate, leading to the formation of dead-end inhibited complexes. In diverse autotrophic organisms this tendency has been counteracted by the recruitment of dedicated AAA+ (ATPases associated with various cellular activities) proteins that all use the energy of ATP hydrolysis to remodel inhibited Rubisco active sites leading to release of the inhibitor. Three evolutionarily distinct classes of these Rubisco activases (Rcas) have been discovered so far. Green and red-type Rca are mostly found in photosynthetic eukaryotes of the green and red plastid lineage respectively, whereas CbbQO is associated with chemoautotrophic bacteria. Ongoing mechanistic studies are elucidating how the various motors are utilizing both similar and contrasting strategies to ultimately perform their common function of cracking the inhibited Rubisco active site. The best studied mechanism utilized by red-type Rca appears to involve transient threading of the Rubisco large subunit C-terminal peptide, reminiscent of the action performed by Clp proteases. As well as providing a fascinating example of convergent molecular evolution, Rca proteins can be considered promising crop-improvement targets. Approaches aiming to replace Rubisco in plants with improved enzymes will need to ensure the presence of a compatible Rca protein. The thermolability of the Rca protein found in crop plants provides an opportunity to fortify photosynthesis against high temperature stress. Photosynthesis also appears to be limited by Rca when light conditions are fluctuating. Synthetic biology strategies aiming to enhance the autotrophic CO2 fixation machinery will need to take into consideration the requirement for Rubisco activases

Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

PubMed

Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

2017-07-01

Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function, and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology, and designing enzymes with new functional capabilities. Proteins 2017; 85:1319-1335. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3′ splice-site organization and activity of U2AF-related proteins

PubMed Central

Kralovicova, Jana; Knut, Marcin; Cross, Nicholas C. P.; Vorechovsky, Igor

2015-01-01

The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3′ splice site (3′ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3′ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3′UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPERα. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing. PMID:25779042

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

PubMed

Kumar, Raj; Calhoun, William J

2008-12-01

Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and

MX Siting Investigation. Water Resources Program Industry Activity Inventory, Nevada-Utah.

DTIC Science & Technology

1980-09-02

sites. New and revived mining activities and the cooling needs of possible new coal -fired electric power plants represent the chief competitors with MX...34 !- ---- ON CO. Figure .-. Ma showing araipce yUaIoto f𔃻XMsieCmlx 1 3 include new mining activity and coal -fired, geothermal, and hydroelectric j energy...in northeastern Juab County. The Soil Conservation Service has been actively pushing land treatment programs to increase the productivity of irrigated

Activation of dioxygen by copper metalloproteins and insights from model complexes

PubMed Central

Quist, David A.; Diaz, Daniel E.; Liu, Jeffrey J.; Karlin, Kenneth D.

2017-01-01

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing met-alloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O2-activation in copper proteins are addressed. PMID:27921179

Modification of the Hemagglutinin Cleavage Site Allows Indirect Activation of Avian Influenza Virus H9N2 by Bacterial Staphylokinase

PubMed Central

Tse, Longping V.; Whittaker, Gary R.

2015-01-01

Influenza H9N2 is considered to be a low pathogenicity avian influenza (LPAI) virus that commonly infects avian species and can also infect humans. In 1996, the influenza virus, A/chicken/Korea/MS96-CE6/1996/H9N2 (MS96) was isolated from an outbreak in multiple farms in South Korea that resulted in upwards of 30% mortality in infected chickens, with the virus infecting a number of extrapulmonary tissues, indicating internal spread. However, in experimental infections, complete recovery of specific pathogen free (SPF) chickens occurred. Such a discrepancy indicated an alternative pathway for MS96 virus to gain virulence in farmed chickens. A key determinant of influenza pathogenesis is the susceptibility of the viral hemagglutinin (HA) to proteolytic cleavage/activation. Here, we identified that an amino acid substitution, Ser to Tyr found at the P2 position of the MS96 HA cleavage site optimizes cleavage by the protease plasmin (Pm). Importantly, we identified that certain Staphylococcus sp. are able to cleave and activate MS96 HA by activating plasminogen (Plg) to plasmin by use of a virulence factor, staphylokinase. Overall, these studies provide an in-vitro mechanism for bacterially mediated enhancement of influenza activation, and allow insight into the microbiological mechanisms underlying the avian influenza H9N2 outbreak in Korea in1996. PMID:25841078

Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase.

PubMed

Zhang, Xiaohua; Caner, Sami; Kwan, Emily; Li, Chunmin; Brayer, Gary D; Withers, Stephen G

2016-11-01

Starch provides the major source of caloric intake in many diets. Cleavage of starch into malto-oligosaccharides in the gut is catalyzed by pancreatic α-amylase. These oligosaccharides are then further cleaved by gut wall α-glucosidases to release glucose, which is absorbed into the bloodstream. Potential surface binding sites for starch on the pancreatic amylase, distinct from the active site of the amylase, have been identified through X-ray crystallographic analyses. The role of these sites in the degradation of both starch granules and soluble starch was probed by the generation of a series of surface variants modified at each site to disrupt binding. Kinetic analysis of the binding and/or cleavage of substrates ranging from simple maltotriosides to soluble starch and insoluble starch granules has allowed evaluation of the potential role of each such surface site. In this way, two key surface binding sites, on the same face as the active site, are identified. One site, containing a pair of aromatic residues, is responsible for attachment to starch granules, while a second site featuring a tryptophan residue around which a malto-oligosaccharide wraps is shown to heavily influence soluble starch binding and hydrolysis. These studies provide insights into the mechanisms by which enzymes tackle the degradation of largely insoluble polymers and also present some new approaches to the interrogation of the binding sites involved.

Enthalpic Breakdown of Water Structure on Protein Active-Site Surfaces

PubMed Central

Haider, Kamran; Wickstrom, Lauren; Ramsey, Steven; Gilson, Michael K.; Kurtzman, Tom

2016-01-01

The principles underlying water reorganization around simple non-polar solutes are well understood and provide the framework for classical hydrophobic effect, whereby water molecules structure themselves around solutes so that they maintain favorable energetic contacts with both the solute and with other water molecules. However, for certain solute surface topographies, water molecules, due to their geometry and size, are unable to simultaneously maintain favorable energetic contacts with both the surface and neighboring water molecules. In this study, we analyze the solvation of ligand-binding sites for six structurally diverse proteins using hydration site analysis and measures of local water structure, in order to identify surfaces at which water molecules are unable to structure themselves in a way that maintains favorable enthalpy relative to bulk water. These surfaces are characterized by a high degree of enclosure, weak solute-water interactions, and surface constraints that induce unfavorable pair interactions between neighboring water molecules. Additionally, we find that the solvation of charged side-chains in an active site generally results in favorable enthalpy but can also lead to pair interactions between neighboring water molecules that are significantly unfavorable relative to bulk water. We find that frustrated local structure can occur not only in apolar and weakly polar pockets, where overall enthalpy tends to be unfavorable, but also in charged pockets, where overall water enthalpy tends to be favorable. The characterization of local water structure in these terms may prove useful for evaluating the displacement of water from diverse protein active-site environments. PMID:27169482

Nevada National Security Site Industrial Sites Project Closeout - 12498

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

Cabble, Kevin; Krauss, Mark; Matthews, Pat

The U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office is responsible for environmental restoration (ER) at the Nevada National Security Site (NNSS). This includes remediation at Industrial Sites where past nuclear testing activities and activities that supported nuclear testing may have or are known to have resulted in the release of contaminants into the environment. Industrial Sites at the NNSS have included nuclear facilities that supported the nuclear rocket/missile development programs, gas stations, landfills, spill sites, ordnance sites, and numerous other waste disposal and release sites. The NNSS Industrial Sites activities neared completion at the endmore » of fiscal year 2011 while other activities required under the Federal Facility Agreement and Consent Order (FFACO) and part of the same NNSS ER Project are forecasted to extend to 2027 or beyond. With the majority of Industrial Sites corrective action units (CAUs) completed (more than 250 CAUs and over 1,800 corrective action sites), it was determined that an activity closeout process should be implemented to ensure that the work completed over the past 15 years is well documented in a comprehensive and concise summary. While the process used to close each individual CAU is described in approved documents, no single document describes in summary fashion the work completed to close the many individual Industrial Sites. The activity closeout process will be used to develop an Industrial Sites closeout document that describes these years of work. This document will summarize the number of Industrial Sites closed under the FFACO and provide general descriptions of projects, contaminants removed, and sites closed in place with corresponding Use Restrictions. Other pertinent information related to Industrial Sites work such as the project history, closure decisions, historical declarations, remediation strategies, and final CAU status will be included in the