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Michael E. Johnson - One of the best experts on this subject based on the ideXlab platform.
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md simulations reveal alternate conformations of the Oxyanion Hole in the zika virus ns2b ns3 protease
Proteins, 2020Co-Authors: Jinhong Ren, Hyun Sun Lee, Alpa Kotak, Michael E. JohnsonAbstract:Recent crystallography studies have shown that the binding site Oxyanion Hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand Oxyanion Hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 310 -helix that corresponds to the inactive conformation of the Oxyanion Hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the Oxyanion Hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and Oxyanion Hole residues. Interestingly, however, transition between the active and inactive conformation of the Oxyanion Hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.
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MD simulations reveal alternate conformations of the Oxyanion Hole in the Zika virus NS2B/NS3 protease.
Proteins: Structure Function and Bioinformatics, 2019Co-Authors: Jinhong Ren, Hyun Sun Lee, Alpa Kotak, Michael E. JohnsonAbstract:Recent crystallography studies have shown that the binding site Oxyanion Hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand Oxyanion Hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 310 -helix that corresponds to the inactive conformation of the Oxyanion Hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the Oxyanion Hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and Oxyanion Hole residues. Interestingly, however, transition between the active and inactive conformation of the Oxyanion Hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.
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design synthesis and evaluation of Oxyanion Hole selective inhibitor substituents for the s1 subsite of factor xa
Bioorganic & Medicinal Chemistry Letters, 2004Co-Authors: Sochanchingwung Rumthao, Debbie C Mulhearn, Wentao Fu, Andrew D Mesecar, Qi Sheng, David Crich, Michael E. JohnsonAbstract:Abstract We have designed, synthesized, and evaluated the factor Xa inhibitory activities of p-amidinophenyl-sulfones, amines, and alcohols intended to take advantage of the polarity and hydrogen-bonding potential of the Oxyanion Hole region of the S1 specificity pocket. We demonstrate that placement of an anionic group within the Oxyanion Hole region of the catalytic site substantially enhances activity, with small flexible groups favored over bulkier ones. Ab initio pKa calculations suggest that the hydroxyl substituent frequently used for benzamidine moieties may be ionized to form an anionic group, consistent with the general trend. One nonamidine based substituent also shows promising activity.
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an Oxyanion Hole selective serine protease inhibitor in complex with trypsin
Bioorganic & Medicinal Chemistry, 2002Co-Authors: Fatima Marankan, Wentao Fu, Andrew D Mesecar, David Crich, Michael E. JohnsonAbstract:p-amidinophenylmethylphosphinic acid (AMPA) was designed, synthesized and crystallized in complex with trypsin to study interactions with the Oxyanion Hole at the S1 site. In comparison to benzamidine, AMPA shows improved activity, which the crystal structure demonstrates to result from hydrogen bonds between the negatively charged phosphinic acid group and the catalytic residues at the Oxyanion Hole.
Rikkert K Wierenga - One of the best experts on this subject based on the ideXlab platform.
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structures of yeast peroxisomal δ3 δ2 enoyl coa isomerase complexed with acyl coa substrate analogues the importance of hydrogen bond networks for the reactivity of the catalytic base and the Oxyanion Hole
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Goodluck U. Onwukwe, Petri M. Pihko, Werner Schmitz, Kristian M Koski, Rikkert K WierengaAbstract:Δ(3),Δ(2)-Enoyl-CoA isomerases (ECIs) catalyze the shift of a double bond from 3Z- or 3E-enoyl-CoA to 2E-enoyl-CoA. ECIs are members of the crotonase superfamily. The crotonase framework is used by many enzymes to catalyze a wide range of reactions on acyl-CoA thioesters. The thioester O atom is bound in a conserved Oxyanion Hole. Here, the mode of binding of acyl-CoA substrate analogues to peroxisomal Saccharomyces cerevisiae ECI (ScECI2) is described. The best defined part of the bound acyl-CoA molecules is the 3',5'-diphosphate-adenosine moiety, which interacts with residues of loop 1 and loop 2, whereas the pantetheine part is the least well defined. The catalytic base, Glu158, is hydrogen-bonded to the Asn101 side chain and is further hydrogen-bonded to the side chain of Arg100 in the apo structure. Arg100 is completely buried in the apo structure and a conformational change of the Arg100 side chain appears to be important for substrate binding and catalysis. The Oxyanion Hole is formed by the NH groups of Ala70 (loop 2) and Leu126 (helix 3). The O atoms of the corresponding peptide units, Gly69 O and Gly125 O, are both part of extensive hydrogen-bond networks. These hydrogen-bond networks are a conserved feature of the crotonase Oxyanion Hole and their importance for catalysis is discussed.
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Structures of yeast peroxisomal Δ3,Δ2‐enoyl‐CoA isomerase complexed with acyl‐CoA substrate analogues: the importance of hydrogen‐bond networks for the reactivity of the catalytic base and the Oxyanion Hole
Acta Crystallographica Section D Biological Crystallography, 2015Co-Authors: Goodluck U. Onwukwe, M. Kristian Koski, Petri M. Pihko, Werner Schmitz, Rikkert K WierengaAbstract:Δ(3),Δ(2)-Enoyl-CoA isomerases (ECIs) catalyze the shift of a double bond from 3Z- or 3E-enoyl-CoA to 2E-enoyl-CoA. ECIs are members of the crotonase superfamily. The crotonase framework is used by many enzymes to catalyze a wide range of reactions on acyl-CoA thioesters. The thioester O atom is bound in a conserved Oxyanion Hole. Here, the mode of binding of acyl-CoA substrate analogues to peroxisomal Saccharomyces cerevisiae ECI (ScECI2) is described. The best defined part of the bound acyl-CoA molecules is the 3',5'-diphosphate-adenosine moiety, which interacts with residues of loop 1 and loop 2, whereas the pantetheine part is the least well defined. The catalytic base, Glu158, is hydrogen-bonded to the Asn101 side chain and is further hydrogen-bonded to the side chain of Arg100 in the apo structure. Arg100 is completely buried in the apo structure and a conformational change of the Arg100 side chain appears to be important for substrate binding and catalysis. The Oxyanion Hole is formed by the NH groups of Ala70 (loop 2) and Leu126 (helix 3). The O atoms of the corresponding peptide units, Gly69 O and Gly125 O, are both part of extensive hydrogen-bond networks. These hydrogen-bond networks are a conserved feature of the crotonase Oxyanion Hole and their importance for catalysis is discussed.
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Structures of yeast peroxisomal Δ(3),Δ(2)-enoyl-CoA isomerase complexed with acyl-CoA substrate analogues: the importance of hydrogen-bond networks for the reactivity of the catalytic base and the Oxyanion Hole.
Acta crystallographica. Section D Biological crystallography, 2015Co-Authors: Goodluck U. Onwukwe, M. Kristian Koski, Petri M. Pihko, Werner Schmitz, Rikkert K WierengaAbstract:Δ(3),Δ(2)-Enoyl-CoA isomerases (ECIs) catalyze the shift of a double bond from 3Z- or 3E-enoyl-CoA to 2E-enoyl-CoA. ECIs are members of the crotonase superfamily. The crotonase framework is used by many enzymes to catalyze a wide range of reactions on acyl-CoA thioesters. The thioester O atom is bound in a conserved Oxyanion Hole. Here, the mode of binding of acyl-CoA substrate analogues to peroxisomal Saccharomyces cerevisiae ECI (ScECI2) is described. The best defined part of the bound acyl-CoA molecules is the 3',5'-diphosphate-adenosine moiety, which interacts with residues of loop 1 and loop 2, whereas the pantetheine part is the least well defined. The catalytic base, Glu158, is hydrogen-bonded to the Asn101 side chain and is further hydrogen-bonded to the side chain of Arg100 in the apo structure. Arg100 is completely buried in the apo structure and a conformational change of the Arg100 side chain appears to be important for substrate binding and catalysis. The Oxyanion Hole is formed by the NH groups of Ala70 (loop 2) and Leu126 (helix 3). The O atoms of the corresponding peptide units, Gly69 O and Gly125 O, are both part of extensive hydrogen-bond networks. These hydrogen-bond networks are a conserved feature of the crotonase Oxyanion Hole and their importance for catalysis is discussed.
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the thiolase reaction mechanism the importance of asn316 and his348 for stabilizing the enolate intermediate of the claisen condensation
Biochemistry, 2009Co-Authors: Gitte Merilainen, Visa Poikela, Petri Kursula, Rikkert K WierengaAbstract:The biosynthetic thiolase catalyzes a Claisen condensation reaction between acetyl-CoA and the enzyme acetylated at Cys89. Two Oxyanion Holes facilitate this catalysis: Oxyanion Hole I stabilizes the enolate intermediate generated from acetyl-CoA, whereas Oxyanion Hole II stabilizes the tetrahedral intermediate of the acetylated enzyme. The latter intermediate is formed when the α-carbanion of acetyl-CoA enolate reacts with the carbonyl carbon of acetyl-Cys89, after which C−C bond formation is completed. Oxyanion Hole II is made of two main chain peptide NH groups, whereas Oxyanion Hole I is formed by a water molecule (Wat82) and NE2(His348). Wat82 is anchored in the active site by an optimal set of hydrogen bonding interactions, including a hydrogen bond to ND2(Asn316). Here, the importance of Asn316 and His348 for catalysis has been studied; in particular, the properties of the N316D, N316A, N316H, H348A, and H348N variants have been determined. For the N316D variant, no activity could be detected. For ...
Jeifu Shaw - One of the best experts on this subject based on the ideXlab platform.
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functional role of catalytic triad and Oxyanion Hole forming residues on enzyme activity of escherichia coli thioesterase i protease i phospholipase l1
Biochemical Journal, 2006Co-Authors: Jeifu ShawAbstract:Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an Oxyanion Hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing Oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the Oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75–80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.
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Crystal Structure of Escherichia coli Thioesterase I/Protease I/Lysophospholipase L1: Consensus Sequence Blocks Constitute the Catalytic Center of SGNH-hydrolases through a Conserved Hydrogen Bond Network
Journal of Molecular Biology, 2003Co-Authors: Su Chang Lin, Jeifu Shaw, Yen Chywan LiawAbstract:Abstract Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing activities of thioesterase, esterase, arylesterase, protease, and lysophospholipase. In particular, TAP has stereoselectivity for amino acid derivative substrates, hence it is useful for the kinetic resolution of racemic mixtures of industrial chemicals. In the present work, the crystal structure of native TAP was determined at 1.9 A, revealing a minimal SGNH-hydrolase fold. The structure of TAP in complex with a diethyl phosphono moiety (DEP) identified its catalytic triad, Ser10-Asp154-His157, and Oxyanion Hole, Ser10-Gly44-Asn73. The Oxyanion Hole of TAP consists of three residues each separated from the other by more than 3.5 A, implying that all of them are highly polarized when substrate bound. The catalytic (His)Ce1–H⋯OC hydrogen bond usually plays a role in the catalytic mechanisms of most serine hydrolases, however, there were none present in SGNH-hydrolases. We propose that the existence of the highly polarized tri-residue-constituted Oxyanion Hole compensates for the lack of a (His)Ce1–H⋯OC hydrogen bond. This suggests that members of the SGNH-hydrolase family may employ a unique catalytic mechanism. In addition, most SGNH-hydrolases have low sequence identities and presently there is no clear criterion to define consensus sequence blocks. Through comparison of TAP and the three SGNH-hydrolase structures currently known, we have identified a unique hydrogen bond network which stabilizes the catalytic center: a newly discovered structural feature of SGNH-hydrolases. We have defined these consensus sequence blocks providing a basis for the sub-classification of SGNH-hydrolases.
Pilar Diaz - One of the best experts on this subject based on the ideXlab platform.
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Rational evolution of the unusual Y-type Oxyanion Hole of Rhodococcus sp. CR53 lipase LipR.
Enzyme and Microbial Technology, 2018Co-Authors: Belén Infanzón, Pablo H. Sotelo, Josefina Martínez, Pilar DiazAbstract:Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/β-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type Oxyanion Hole, never found before among bacterial lipases. In order to explore this unusual Y-type Oxyanion Hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type Oxyanion Hole (YDS) into one closer to those most frequently found in bacteria (GGG(X)). However, activity was completely lost in all mutants obtained, indicating that the Y-type Oxyanion Hole of LipR is required for activity. A second approach was addressed to modify the two main Oxyanion Hole residues Tyr110 and Asp111, previously described for CAL-A as the most relevant amino acids involved in stabilization of the enzyme-substrate complex. A saturation mutagenesis library was prepared for each residue (Tyr110 and Asp111), and activity of the resulting variants was assayed on different chain length substrates. No functional LipR variants could be obtained when Tyr110 was replaced by any other amino acids, indicating that this is a crucial residue for catalysis. However, among the Asp111 variants obtained, LipR D111G produced a functional enzyme. Interestingly, this LipR-YGS variant showed less activity than wild type LipR on short- or mid- chain substrates but displayed a 5.6-fold increased activity on long chain length substrates. Analysis of the 3D model and in silico docking studies of this enzyme variant suggest that substitution of Asp by Gly produces a wider entrance tunnel that would allow for a better and tight accommodation of larger substrates, thus justifying the experimental results obtained.
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Improving enantioselectivity towards tertiary alcohols using mutants of Bacillus sp. BP-7 esterase EstBP7 holding a rare GGG(X)-Oxyanion Hole
Applied Microbiology and Biotechnology, 2014Co-Authors: Amanda Fillat, Pedro Romea, Fèlix Urpí, F. I. Javier Pastor, Pilar DiazAbstract:Lipases and esterases are important biocatalysts for synthetic organic fine chemistry. An esterase from Bacillus sp. BP-7 (EstBP7) bears in its amino acid sequence a rare GGG(A)X Oxyanion Hole motif, where an uncommon threonine (T) is found at the third position. Detection of this pattern motivated evaluation of the ability of EstBP7 for conversion of tertiary alcohols. The enzyme was engineered in order to optimize its performance to provide important chiral building blocks: five variants with mutations in the Oxyanion Hole motif were created to investigate the influence on activity and enantioselectivity in the kinetic resolution of eight acetates of tertiary alcohols. Wild-type enzyme converted all esters of tertiary alcohols assayed with low enantioselectivity, whereas some of the mutants displayed significantly increased E-values. One of the mutants (EstBP7-AGA; Mut 5) showed an E >100 towards a complex tertiary alcohol acetate (2-(4-pyridyl)but-3-yn-2-yl acetate) at low reaction temperature (4 °C). Therefore, the catalytic toolbox was expanded for biocatalysis of optically pure tertiary alcohols valuable for the pharmaceutical industry.
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Unusual carboxylesterase bearing a GGG(A)X-type Oxyanion Hole discovered in Paenibacillus barcinonensis BP-23
Biochimie, 2014Co-Authors: Belén Infanzón, Amanda Fillat, F. I. Javier Pastor, Susana V. Valenzuela, Pilar DiazAbstract:Strain Paenibacillus barcinonensis BP-23, previously isolated from Ebro's river delta (Spain), bears a complex hydrolytic system showing the presence of at least two enzymes with activity on lipidic substrates. EstA, a cell-bound B-type carboxylesterase from the strain was previously isolated and characterized. The gene coding for a second putative lipase, located upstream cellulase Cel5A, was obtained using a genome walking strategy and cloned in Escherichia coli for further characterization. The recombinant clone obtained displayed high activity on medium/short-chain fatty acid-derivative substrates. The enzyme, named Est23, was purified and characterized, showing maximum activity on pNP-caprylate (C8:0) or MUF-heptanoate (C7:0) under conditions of moderate temperature and pH. Although Est23 displays a GGG(A)X-type Oxyanion Hole, described as an important motif for tertiary alcohol ester resolution, neither conversion nor enantiomeric resolution of tertiary alcohols could be detected. Amino acid sequence alignment of Est23 with those of known bacterial lipase families and with closely related proteins suggests that the cloned enzyme does not belong to any of the described bacterial lipase families. A phylogenetic tree including Est23 and similar amino acid sequences showed that the enzyme belongs to a differentiated sequence cluster which probably constitutes a new family of bacterial lipolytic enzymes.
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Special Rhodococcus sp. CR-53 esterase Est4 contains a GGG(A)X-Oxyanion Hole conferring activity for the kinetic resolution of tertiary alcohols.
Applied Microbiology and Biotechnology, 2013Co-Authors: Arnau Bassegoda, F. I. Javier Pastor, Amanda Fillat, Pilar DiazAbstract:Rhodococci are highly adaptable bacteria, capable to degrade or transform a large number of organic compounds, including recalcitrant or toxic products. However, little information is available on the lipases of the genus Rhodococcus, except for LipR, the first lipase isolated and described from strain Rhodococcus CR-53. Taking into consideration the interest raised by the enzymes produced by actinomycetes, a search for new putative lipases was performed in strain Rhodococcus CR-53. We describe here the isolation, cloning, and characterization of intracellular esterase Est4, a mesophilic enzyme showing preference for short-chain-length acyl groups, without interfacial activation. Est4 displays moderate thermal and pH stability and low tolerance to most tested ions, being inhibited by detergents like sodium dodecyl sulfate and Triton X-100®. Nevertheless, the enzyme shows good long-term stability when stored at 4–20 °C and neutral pH. Amino acid sequence analysis of Est4 revealed a protein of 313 amino acids without a signal peptide, bearing most of the conserved blocks that define bacterial lipase family IV, thus being assigned to this family. Detection of a GGG(A)X Oxyanion Hole in the enzyme motivated the evaluation of Est4 ability to convert tertiary alcohol esters. The newly discovered esterase Est4 from Rhodococcus CR-53 successfully hydrolyzed the tertiary alcohol esters linalyl acetate, terpinyl acetate, and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate.
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rhodococcus sp strain cr 53 lipr the first member of a new bacterial lipase family family x displaying an unusual y type Oxyanion Hole similar to the candida antarctica lipase clan
Applied and Environmental Microbiology, 2012Co-Authors: Arnau Bassegoda, F Javier I Pastor, Pilar DiazAbstract:ABSTRACT Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type Oxyanion Hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.
Kaillathe Padmanabhan - One of the best experts on this subject based on the ideXlab platform.
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high resolution structures of p aminobenzamidine and benzamidine viia soluble tissue factor unpredicted conformation of the 192 193 peptide bond and mapping of ca2 mg2 na and zn2 sites in factor viia
Journal of Biological Chemistry, 2006Co-Authors: Paul S Bajaj, Amy E Schmidt, Sayeh Agah, Madhu S Bajaj, Kaillathe PadmanabhanAbstract:Abstract Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the Oxyanion Hole in this structure. Importantly, the Oxyanion Hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and Oxyanion Hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces Oxyanion Hole formation and functional active site geometry in FVIIa. Absence of Oxyanion Hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.
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High Resolution Structures of p-Aminobenzamidine- and Benzamidine-VIIa/Soluble Tissue Factor: Unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+ and Zn2+ sites in factor VIIa
Journal of Biological Chemistry, 2006Co-Authors: S. Paul Bajaj, Amy E Schmidt, Sayeh Agah, Madhu S Bajaj, Kaillathe PadmanabhanAbstract:Abstract Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the Oxyanion Hole in this structure. Importantly, the Oxyanion Hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and Oxyanion Hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces Oxyanion Hole formation and functional active site geometry in FVIIa. Absence of Oxyanion Hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.
