scholarly journals Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila

2006 ◽  
Vol 188 (3) ◽  
pp. 1143-1154 ◽  
Author(s):  
Sarah H. Lawrence ◽  
Kelvin B. Luther ◽  
Hermann Schindelin ◽  
James G. Ferry
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Van Der Waals Interactions ◽  
Nucleophilic Attack ◽  
Titration Calorimetry ◽  
Acetyl Phosphate ◽  
Methanosarcina Thermophila ◽  
Functional Studies ◽  
Active Site Cleft

ABSTRACT Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD ], 20 μM) and low-affinity (KD , 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

2016 ◽  
Vol 72 (6) ◽  
pp. 490-499
Author(s):  
Mirja Krause ◽  
Tiila-Riikka Kiema ◽  
Peter Neubauer ◽  
Rik K. Wierenga
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Directed Evolution ◽  
Crystal Structures ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Point Mutations ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

scholarly journals Structure of hydrogen sulfide-producing enzyme from a periodontal pathogen

2014 ◽  
Vol 70 (a1) ◽  
pp. C454-C454
Author(s):  
Yuichiro Kezuka ◽  
Yasuo Yoshida ◽  
Takamasa Nonaka
Keyword(s):  
Hydrogen Sulfide ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Intermediate Formation ◽  
Fusobacterium Nucleatum ◽  
Oral Bacteria ◽  
Periodontal Pathogen ◽  
Fold Axis ◽  
Key Species ◽  
Active Site Cleft

Hydrogen sulfide (H2S) is one of the predominant volatile sulfur compounds that are primarily responsible for oral malodor and contribute to the progress of periodontal disease. H2S in the human oral cavity is generally produced by enzymatic actions of oral bacteria.Fusobacterium nucleatum, a Gram negative periodontal pathogen, is known to be one of the heaviest H2S producers [1]. For now, four genes (fn0625,fn1055,fn1220, andfn1419) encoding pyridoxal-5′-phosphate (PLP)-dependent H2S-producing enzymes have been identified and characterized inF. nucleatumATCC 25586. Of the four enzymes, Fn1055 protein is a unique H2S-producing enzyme, which produces H2S and L-serine from L-cysteine [2]. Therefore, Fn1055 might play important roles in L-serine biosynthesis in addition to H2S production in this periodontal pathogen. Crystal structures of recombinant Fn1055 and its site-directed mutant complex with L-cysteine (a substrate) were determined at 2.1 Å resolution. The enzyme forms a homodimer whose subunits are related by a two-fold axis. The subunit is composed of two domains with α/β structure. The PLP cofactor forms a covalent internal aldimine linkage with the ε-amino group of Lys46 at the bottom of active site cleft between the domains, in the absence of substrate. On the other hand, in the cocrystal of mutant with L-cysteine, the introduced L-cysteine was found to be covalently bound to PLP, instead of Lys46. This covalent intermediate was identified as an α-aminoacrylate, which is the key species of PLP-dependent-enzyme catalysis, by spectrophotometric measurement. Along with the intermediate formation, closure of active site cleft was also observed. Furthermore, we found an amino acid residue acting as a base and confirmed its indispensability for catalysis by enzymatic analyses. These results support that H2S production by Fn1055 proceeds through the β-elimination of L-cysteine, and enable us to propose a detailed catalytic mechanism of Fn1055.

Natural inhibitors of thrombin

2014 ◽  
Vol 111 (04) ◽  
pp. 583-589 ◽  
Author(s):  
James Huntington
Keyword(s):  
Blood Flow ◽  
Crystal Structures ◽  
Active Site ◽  
Anion Binding ◽  
Thrombin Inhibitors ◽  
Multiple Functions ◽  
Endogenous Inhibitors ◽  
Active Site Cleft ◽  
Single Protein ◽  
Natural Inhibitors

SummaryThe serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

scholarly journals A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

2016 ◽  
Vol 113 (8) ◽  
pp. 2068-2073 ◽  
Author(s):  
Erik W. Debler ◽  
Kanishk Jain ◽  
Rebeccah A. Warmack ◽  
You Feng ◽  
Steven G. Clarke ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Type I ◽  
Histone H4 ◽  
Active Site Residues ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Product Specificity

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

scholarly journals Structure of Allium lachrymatory factor synthase elucidates catalysis on sulfenic acid substrate

2017 ◽  
Author(s):  
Takatoshi Arakawa ◽  
Yuta Sato ◽  
Jumpei Takabe ◽  
Noriya Masamura ◽  
Masahiro Kato ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Sigmatropic Rearrangement ◽  
Natural Production ◽  
Lachrymatory Factor ◽  
Biological Decomposition ◽  
Key Residues ◽  
Lachrymatory Factor Synthase

AbstractNatural lachrymatory effects are invoked by small volatile S-oxide compounds. They are produced through alkene sulfenic acids by the action of lachrymatory factor synthase (LFS). Here we present the crystal structures of onion LFS (AcLFS) revealed in solute-free and two solute-stabilized forms. Each structure adopts a single seven-stranded helix-grip fold possessing an internal pocket. Mutagenesis analysis localized the active site to a layer near the bottom of the pocket, which is adjacent to the deduced key residues Arg71, Glu88, and Tyr114. Solute molecules visible on the active site have suggested that AcLFS accepts various small alcohol compounds as well as its natural substrate, and they inhibit this substrate according to their chemistry. Structural homologs have been found in the SRPBCC superfamily, and comparison of the active sites has demonstrated that the electrostatic potential unique to AcLFS could work in capturing the substrate in its specific state. Finally, we propose a rational catalytic mechanism based on intramolecular proton shuttling in which the microenvironment of AcLFS can bypass the canonical [1,4]-sigmatropic rearrangement principle established by microwave studies. Beyond revealing how AcLFS generates the lachrymatory compound, this study provides insights into the molecular machinery dealing with highly labile organosulfur species.Significance statementCrushing of onion liberates a volatile compound, syn-propanethial S-oxide (PTSO), which causes lachrymatory effect on humans. We present the crystal structures of onion LFS (AcLFS), the enzyme responsible for natural production of PTSO. AcLFS features a barrel-like fold, and mutagenic and inhibitory analyses revealed that the key residues are present in the central pocket, harboring highly concentrated aromatic residues plus a dyad motif. The architecture of AcLFS is widespread among proteins with various biological functions, such as abscisic acid receptors and polyketide cyclases, and comparisons with these homologs indicate that unique steric and electronic properties maintain the pocket as a reaction compartment. We propose the molecular mechanism behind PTSO generation and shed light on biological decomposition of short-lived sulfur species.

scholarly journals Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Martino L. Di Salvo ◽  
J. Neel Scarsdale ◽  
Galina Kazanina ◽  
Roberto Contestabile ◽  
Verne Schirch ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Serine Hydroxymethyltransferase ◽  
Active Site Residue ◽  
Wild Type ◽  
Hydroxymethyl Group ◽  
New Energy ◽  
Mutant Forms

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stablegem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion ofgem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and theε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

scholarly journals Crystal structures of glutathione- and inhibitor-bound human GGT1: Critical interactions within the cysteinylglycine binding site

2020 ◽  
pp. jbc.RA120.016265
Author(s):  
Simon S. Terzyan ◽  
Luong T. Nguyen ◽  
Anthony W.G. Burgett ◽  
Annie Heroux ◽  
Clyde A Smith ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Crystal Structures ◽  
Active Site ◽  
Molecular Level ◽  
Amino Acid Residues ◽  
Active Site Cleft ◽  
Substrate Cleavage ◽  
Glutamyl Transpeptidase ◽  
Specific Inhibitors

Overexpression of γ-glutamyl transpeptidase(GGT1) has been implicated in an array of humandiseases including asthma, reperfusion injury,and cancer. Inhibitors are needed for therapy, butdevelopment of potent, specific inhibitors ofGGT1 has been hampered by a lack of structuralinformation regarding substrate binding andcleavage. To enhance our understanding of themolecular mechanism of substrate cleavage, wehave solved the crystal structures of humanGGT1 (hGGT1) with glutathione (a substrate)and a phosphate-glutathione analog (anirreversible inhibitor) bound in the active site.These are the first structures of any eukaryoticGGT with the cysteinylglycine region of thesubstrate-binding site occupied. These structuresand the structure of apo-hGGT reveal movementof amino acid residues within the active site as thesubstrate binds. Asn-401 and Thr-381 each formhydrogen bonds with two atoms of GSH spanningthe γ-glutamyl bond. Three different atoms ofhGGT1 interact with the carboxyl-oxygen of thecysteine of GSH. Interactions between theenzyme and substrate change as the substratemoves deeper into the active site cleft. Thesubstrate reorients and a new hydrogen bond isformed between the substrate and the oxyanionhole. Thr-381 is locked into a singleconformation as an acyl bond forms between thesubstrate and the enzyme. These data provideinsight on a molecular level into the substratespecificity of hGGT1 and provide an explanationfor seemingly disparate observations regardingthe enzymatic activity of hGGT1 mutants. Thisknowledge will aid in the design of clinicallyuseful hGGT1 inhibitors.

Sign in / Sign up

Export Citation Format

Share Document