Alpha/beta‐hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families

The alpha/beta‐hydrolases are a family of acid‐base‐nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn. We show that binding of an external ligand does not change the structure of the catalytic acid zone and both the ligand‐free and ligand‐bound forms of the protein belong to the same catalytic acid zone subgroup. We also show that the catalytic acid zone coordinates the position of the catalytic histidine loop directly above its plane, and consequently, fixes the catalytic histidine in a proper position near the catalytic acid. Finally, we demonstrate that the catalytic acid zone plays a key role in multi‐subunit complex formation in ABH enzymes, and is involved in interactions with other proteins. As a result, we speculate that each of the catalytic triad residues has its own supporting structural scaffold, similar to the catalytic acid zone, described above, which together form the extended catalytic triad motif. Each scaffold coordinates the function of its respective catalytic residue, and can even compensate for the loss of protein function, if the catalytic amino acid is mutated.     

New insight into the architecture of oxy‐anion pocket in unliganded conformation of GAT domains: A MD‐simulation study

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N‐terminal “glutaminase” (GAT) and C‐terminal “synthetase” domain. The enzyme is identified as a potential target for anti‐cancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H‐bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)‐substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non‐catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site. Proteins 2016; 84:360–373. © 2016 Wiley Periodicals, Inc.     

Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Intense efforts are underway to identify inhibitors of the enzyme gamma‐glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma‐glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6‐diazo‐5‐oxo‐norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM ^?1 min^?1 and the K _i was 2.7 ± 0.7 mM . The crystal structure of DON‐inactivated hGGT1 contained a molecule of DON without the diazo‐nitrogen atoms in the active site. The overall structure of the hGGT1‐DON complex resembled the structure of the apo‐enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1‐DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α‐amine of Thr381. The structure of DON‐bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.     

High resolution crystal structures of human cytosolic thiolase (CT): a comparison of the active sites of human CT, bacterial thiolase, and bacterial KAS I

Thiolases belong to a superfamily of condensing enzymes that includes also beta-ketoacyl acyl carrier protein synthases (KAS enzymes), involved in fatty acid synthesis. Here, we describe the high resolution structure of human cytosolic acetoacetyl-CoA thiolase (CT), both unliganded (at 2.3 angstroms resolution) and in complex with CoA (at 1.6 angstroms resolution). CT catalyses the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to the synthesis of cholesterol. CT is a homotetramer of exact 222 symmetry. There is an excess of positively charged residues at the interdimer surface leading towards the CoA-binding pocket, possibly important for the efficient capture of substrates. The geometry of the catalytic site, including the three catalytic residues Cys92, His 353, Cys383, and the two oxyanion holes, is highly conserved between the human and bacterial Zoogloea ramigera thiolase. In human CT, the first oxyanion hole is formed by Wat38 (stabilised by Asn321) and NE2(His353), and the second by N(Cys92) and N(Gly385). The active site of this superfamily is constructed on top of four active site loops, near Cys92, Asn321, His353, and Cys383, respectively. These loops were used for the superpositioning of CT on the bacterial thiolase and on the Escherichia coli KAS I. This comparison indicates that the two thiolase oxyanion holes also exist in KAS I at topologically equivalent positions. Interestingly, the hydrogen bonding interactions at the first oxyanion hole are different in thiolase and KAS I. In KAS I, the hydrogen bonding partners are two histidine NE2 atoms, instead of a water and a NE2 side-chain atom in thiolase. The second oxyanion hole is in both structures shaped by corresponding main chain peptide NH-groups. The possible importance of bound water molecules at the catalytic site of thiolase for the reaction mechanism is discussed.     

Crystallographic analysis of a thermoactive nitrilase

The nitrilase superfamily is a large and diverse superfamily of enzymes that catalyse the cleavage of various types of carbon-nitrogen bonds using a Cys-Glu-Lys catalytic triad. Thermoactive nitrilase from Pyrococcus abyssi (PaNit) hydrolyses small aliphatic nitriles like fumaro- and malononitryl. Yet, the biological role of this enzyme is unknown. We have analysed several crystal structures of PaNit: without ligands, with an acetate ion bound in the active site and with a bromide ion in the active site. In addition, docking calculations have been performed for fumaro- and malononitriles. The structures provide a proof for specific binding of the carboxylate ion and a general affinity for negatively changed ligands. The role of residues in the active site is considered and an enzymatic reaction mechanism is proposed in which Cys146 acts as the nucleophile, Glu42 as the general base, Lys113/Glu42 as the general acid, WatA as the hydrolytic water and Nζ_Lys113 and N_Phe147 form the oxyanion hole.     

Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation

The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a large-scale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis.     

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole

Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu²²⁴ and the side chain of Arg²³³ form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.     

Molecular Anatomy of Tyrosinase and its Related Proteins: Beyond the Histidine‐Bound Metal Catalytic Center

The structure of tyrosinase (Tyr) is reviewed from a double point of view. On the one hand, by comparison of all Tyr found throughout nature, from prokaryotic organisms to mammals and on the other, by comparison with the tyrosinase related proteins (Tyrps) that appeared late in evolution, and are only found in higher animals. Their structures are reviewed as a whole rather than focused on the histidine (His)‐bound metal active site, which is the part of the molecule common to all these proteins. The availability of crystallographic data of hemocyanins and recently of sweet potato catechol oxidase has improved the model of the three‐dimensional structure of the Tyr family. Accordingly, Tyr has a higher structural disorder than hemocyanins, particularly at the CuA site. The active site seems to be characterized by the formation of a hydrophobic pocket with a number of conserved aromatic residues sited close to the well‐known His. Other regions specific of the mammalian enzymes, such as the cytosolic C‐terminal tail, the cysteine clusters, and the N‐glycosylation sequons, are also discussed. The complete understanding of the Tyr copper‐binding domain and the characterization of the residues determinant of the relative substrate affinities of the Tyrps will improve the design of targeted mutagenesis experiments to understand the different catalytic capabilities of Tyr and Tyrps. This may assist future aims, from the design of more efficient bacterial Tyr for biotechnological applications to the design of inhibitors of undesirable fruit browning in vegetables or of color skin modulators in animals.     

Combined docking and molecular dynamics simulations to enlighten the capacity of Pseudomonas cepacia and Candida antarctica lipases to catalyze quercetin acetylation

A combined docking and molecular dynamics protocol was applied to investigate quercetin binding modes within the catalytic cavity of Candida antarctica lipase B (CALB) and Pseudomonas cepacia lipase (PCL), aiming to explain the difference of specificity of these enzymes in acetylation reaction. For both lipases, docking of quercetin yielded two families of conformers with either the quercetin A or B-ring pointing towards the catalytic residues. Molecular dynamics (MD) calculations were subsequently performed on several complexes of each family. MD trajectories were analyzed focusing on the orientation of the acyl donor bound to the catalytic serine towards the oxyanion hole residues and the proximity of quercetin hydroxyl groups to the catalytic residues. Results showed that with CALB, the acetate was not correctly positioned within the oxyanion hole whatever the orientation of quercetin, suggesting that no product could be obtained. With PCL, the acetate remained within the oxyanion hole during all MD trajectories. Depending on quercetin orientation, either the 7-OH group or the 3, 5, 3′, 4′-OH groups came alternatively near the catalytic residues, suggesting that all of them could be acylated. The capacity of models to explain the regioselectivity of the reaction was discussed. Key residues and interactions involved in quercetin binding modes were identified and related to the reaction feasibility.     

Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.     

X‐ray crystallographic structure of BshB,the zinc‐dependent deacetylase involved in bacillithiol biosynthesis

Many gram‐positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three‐enzyme pathway that includes the action of the zinc‐dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X‐ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.     

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.     

Crystal structure and enhanced activity of a cutinase‐like enzyme from Cryptococcus sp. strain S‐2

The structural and enzymatic characteristics of a cutinase‐like enzyme (CLE) from Cryptococcus sp. strain S‐2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 Å resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long‐chain substrates, as well as the preferred specificity of FS cutinase for short‐chain substrates. In addition, site‐directed mutagenesis was performed to increase the hydrolysis activity on long‐chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long‐chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Molecular characterization of novel pyridoxal‐5′‐phosphate‐dependent enzymes from the human microbiome

Pyridoxal‐5′‐phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP‐dependent enzymes account for ～1.5% of most prokaryotic genomes and are estimated to be involved in ～4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP‐dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram‐positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP‐dependent enzyme fold and structure‐guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.     

Structural comparison of Ntn-hydrolases

The Ntn-hydrolases (N-terminal nucleophile) are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N-terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is almost completely absent, the enzymes share a similar alphabeta betaalpha-core structure. The central beta-sheets in the core were found to have different packing angles, ranging from 5 to 35 degrees. In the Ntn-hydrolases under study, eight totally conserved secondary structure units were found (region C). Five of them were observed to contain the greatest number of conserved and functionally important residues and are therefore crucial for the structure and function of Ntn-hydrolases. Two additional regions, consisting of secondary structure units (regions A and B), were found to be in structurally similar locations, but in different orders in the polypeptide chain. The catalytic machinery is located in the structures in a similar manner, and thus the catalytic mechanisms of all of the enzymes are probably similar. However, the substrate binding and the oxyanion hole differed partially.     

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.     

Glycoside hydrolases: Catalytic base/nucleophile diversity

Recent studies have shown that a number of glycoside hydrolase families do not follow the classical catalytic mechanisms, as they lack a typical catalytic base/nucleophile. A variety of mechanisms are used to replace this function, including substrate‐assisted catalysis, a network of several residues, and the use of non‐carboxylate residues or exogenous nucleophiles. Removal of the catalytic base/nucleophile by mutation can have a profound impact on substrate specificity, producing enzymes with completely new functions. Biotechnol. Bioeng. 2010;107: 195–205. © 2010 Wiley Periodicals, Inc.     

Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase

Allosteric communication between different subunits in metabolic enzyme complexes is of utmost physiological importance but only understood for few systems. We analyzed the structural basis of allostery in aminodeoxychorismate synthase (ADCS), which is a member of the family of glutamine amidotransferases and catalyzes the committed step of the folate biosynthetic pathway. ADCS consists of the synthase subunit PabB and the glutaminase subunit PabA, which is allosterically stimulated by the presence of the PabB substrate chorismate. We first solved the crystal structure of a PabA subunit at 1.9-Å resolution. Based on this structure and the known structure of PabB, we computed an atomic model for the ADCS complex. We then used alanine scanning to test the functional role of 59 conserved residues located between the active sites of PabB and PabA. Steady-state kinetic characterization revealed four branches of a conserved network of mainly charged residues that propagate the signal from chorismate at the PabB active site to the PabA active site. The branches eventually lead to activity-inducing transformations at (i) the oxyanion hole motif, (ii) the catalytic Cys‐His‐Glu triad, and (iii) glutamine binding residues at the PabA active site. We compare our findings with previously postulated activation mechanisms of different glutamine amidotransferases and propose a unifying regulation mechanism for this ubiquitous family of enzymes.     

Structural characterization of aminoglycoside 4′‐O‐adenylyltransferase ANT(4′)‐IIb from Pseudomonas aeruginosa

Aminoglycosides were one of the first classes of broad‐spectrum antibacterial drugs clinically used to effectively combat infections. The rise of resistance to these drugs, mediated by enzymatic modification, has since compromised their utility as a treatment option, prompting intensive research into the molecular function of resistance enzymes. Here, we report the crystal structure of aminoglycoside nucleotidyltransferase ANT(4′)‐IIb in apo and tobramycin‐bound forms at a resolution of 1.6 and 2.15 Å, respectively. ANT(4′)‐IIb was discovered in the opportunistic pathogen Pseudomonas aeruginosa and conferred resistance to amikacin and tobramycin. Analysis of the ANT(4′)‐IIb structures revealed a two‐domain organization featuring a mixed β‐sheet and an α‐helical bundle. ANT(4′)‐IIb monomers form a dimer required for its enzymatic activity, as coordination of the aminoglycoside substrate relies on residues contributed by both monomers. Despite harbouring appreciable primary sequence diversity compared to previously characterized homologues, the ANT(4′)‐IIb structure demonstrates a surprising level of structural conservation highlighting the high plasticity of this general protein fold. Site‐directed mutagenesis of active site residues and kinetic analysis provides support for a catalytic mechanism similar to those of other nucleotidyltransferases. Using the molecular insights provided into this ANT(4′)‐IIb‐represented enzymatic group, we provide a hypothesis for the potential evolutionary origin of these aminoglycoside resistance determinants.     

The structural basis for catalysis and substrate specificity of a rhomboid protease

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism‐based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S₁‐ and S₂′‐binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor‐bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.