Crystal Structures of Substrates and Products Bound to the Phosphoglycerate Kinase Active Site Reveal the Catalytic Mechanism†

Biochemistry ◽  
1998 ◽  
Vol 37 (13) ◽  
pp. 4429-4436 ◽  
Author(s):  
Bradley E. Bernstein ◽  
Wim G. J. Hol
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Phosphoglycerate Kinase

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 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 Structures of theMycobacterium tuberculosisGlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

2018 ◽  
Vol 74 (4) ◽  
pp. 321-331
Author(s):  
Nina M. Wolf ◽  
Hiten J. Gutka ◽  
Farahnaz Movahedzadeh ◽  
Celerino Abad-Zapatero
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Adenosine Monophosphate ◽  
Class Ii ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Oligomeric State ◽  
Fructose 1,6 Bisphosphatase ◽  
Lead Inhibitors

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) fromMycobacterium tuberculosisat 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase fromSynechocystis(strain 6803) as well as the equivalent enzyme fromThermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII fromEscherichia coliand is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII fromM. tuberculosis(MtFBPaseII) is conserved and is analogous to that described previously for theE. colienzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures ofMtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for theSynechocystisenzyme. The structural and functional insights derived from the structure ofMtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

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.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias Zeug ◽  
Nebojsa Markovic ◽  
Cristina V. Iancu ◽  
Joanna Tripp ◽  
Mislav Oreb ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Enzyme Engineering ◽  
Base Catalysis ◽  
Oxidative Decarboxylation ◽  
Transition State Stabilization ◽  
Major Bottleneck ◽  
Hydroxybenzoic Acids ◽  
Potassium Binding ◽  
Β Sheet

AbstractHydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

scholarly journals Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Riley B. Peacock ◽  
Taylor McGrann ◽  
Marco Tonelli ◽  
Elizabeth A. Komives
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Protein C ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Exchange Mass Spectrometry ◽  
Catalytically Active ◽  
Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

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