Enzymes in the glutathione transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to electrophilic substrates. As a consequence they are involved in a number of key biological processes, including protection of cells against chemical damage, steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. Although virtual screening has been used widely to discover substrates by docking potential noncovalent ligands into active site clefts of enzymes, docking has been rarely constrained by a covalent bond between the enzyme and ligand. In this study, we investigate the accuracy of docking poses and substrate discovery in the GST superfamily, by docking 6738 potential ligands from the KEGG and MetaCyc compound libraries into 14 representative GST enzymes with known structures and substrates using the PLOP program [Jacobson Proteins 2004, 55, 351]. For X-ray structures as receptors, one of the top 3 ranked models is within 3 Å all-atom root mean square deviation (RMSD) of the native complex in 11 of the 14 cases; the enrichment LogAUC value is better than random in all cases, and better than 25 in 7 of 11 cases. For comparative models as receptors, near-native ligand–enzyme configurations are often sampled but difficult to rank highly. For models based on templates with the highest sequence identity, the enrichment LogAUC is better than 25 in 5 of 11 cases, not significantly different from the crystal structures. In conclusion, we show that covalent docking can be a useful tool for substrate discovery and point out specific challenges for future method improvement.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn2+-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosBSa) has been determined to a resolution of 1.15 Å. Cocrystallization of FosBSa with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosBSa contain Zn2+ in the active site, kinetic analyses of FosBSa indicated that the enzyme is inhibited by Zn2+ for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M2+-dependent thiol tranferases that catalyze nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosBBc). The overall structure of FosBBc, at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosBBc cocrystallized with fosfomycin and a variety of divalent metals, including Ni2+, Mn2+, Co2+, and Zn2+, indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosBBc structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate l-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosBBc in complex with the l-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn2+ metal in the active site have been determined. The l-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosBBc indicated that the enzyme has a preference for BSH over l-Cys when activated by Mn2+ and is inhibited by Zn2+. The fact that Zn2+ is an inhibitor of FosBBc was used to obtain a ternary complex structure of the enzyme with both fosfomycin and l-Cys bound.

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, d-glucosamine hydrochloride, in 11 steps and 8–9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with FosB from Bacillus subtilis, Bacillus anthracis, or Bacillus cereus.

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E2 (PGE2). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a naturally occurring degradation product of prostaglandin D2, is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ2 can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH2) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ2 were determined. The question of whether the anti-inflammatory properties of 15d-PGJ2 are due to inhibition of MPGES1 is discussed.

The inducible microsomal prostaglandin E2 synthase 1 (MPGES1) is an integral membrane protein coexpressed with and functionally coupled to cyclooxygenase 2 (COX-2) generating the pro-inflammatory molecule PGE2. The development of effective inhibitors of MPGES1 holds promise as a highly selective route for controlling inflammation. In this paper, we describe the use of backbone amide H/D exchange mass spectrometry to map the binding sites of different types of inhibitors of MPGES1. The results reveal the locations of specific inhibitor binding sites that include the GSH binding site and a hydrophobic cleft in the protein thought to accommodate the prostaglandin H2 substrate. In the absence of three-dimensional crystal structures of the enzyme-bound inhibitors, the results provide clear physical evidence that three pharmacologically active inhibitors bind in a hydrophobic cleft composed of sections of transmembrane helices Ia, IIb, IIIb, and IVb at the interface of subunits in the trimer. In principle, the H/D exchange behavior of the protein can be used as a preliminary guide for optimization of inhibitor efficacy. Finally, a comparison of the structures and H/D exchange behavior of MPGES1 and the related enzyme MGST1 in the presence of glutathione and the inhibitor glutathione sulfonate confirms the unusual observation that two proteins from the same superfamily harbor GSH binding sites in different locations.

The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (Kd1 = 0.07 ± 0.03 mM) and one weak (Kd2 = 1.3 ± 0.2 mM) binding site for GSH. YghU exhibits little or no GSH transferase activity with most typical electrophilic substrates but does possess a modest catalytic activity toward several organic hydroperoxides. Most notably, the enzyme also exhibits disulfide-bond reductase activity toward 2-hydroxyethyl disulfide [kcat = 74 ± 6 s−1, and kcat/KMGSH = (6.6 ± 1.3) × 104 M−1 s−1] that is comparable to that previously determined for YfcG. A superposition of the structures of the YghU·2GSH and YfcG·GSSG complexes reveals a remarkable structural similarity of the active sites and the 2GSH and GSSG molecules in each. We conclude that the two structures represent reduced and oxidized forms of GSH-dependent disulfide-bond oxidoreductases that are distantly related to glutaredoxin 2. The structures and properties of YghU and YfcG indicate that they are members of the same, but previously unidentified, subfamily of GSH transferase homologues, which we suggest be called the nu-class GSH transferases.

YfcG is one of eight glutathione (GSH) transferase homologues encoded in the Escherichia coli genome. The protein exhibits low or no GSH transferase activity toward a panel of electrophilic substrates. In contrast, it has a very robust disulfide-bond reductase activity toward 2-hydroxyethyldisulfide on par with mammalian and bacterial glutaredoxins. The structure of YfcG at 2.3 Å-resolution from crystals grown in the presence of GSH reveals a molecule of glutathione disulfide in the active site. The crystallographic results and the lack of functional cysteine residues in the active site of YfcG suggests that the reductase activity is unique in that no sulfhydryl groups in the YfcG protein are covalently involved in the redox chemistry.

The fosfomycin (1) resistance proteins FosA and FosX in pathogenic microorganisms are related to a catalytically promiscuous progenitor encoded in a phn operon in Mesorhizobium loti. The mlr3345 gene product (FosXMl) from M. loti has a very low epoxide hydrolase activity and even lower glutathione transferase activity toward 1 and does not confer resistance to the antibiotic. In vitro homologous recombination of the mlr3345 and pa1129 genes (a fosA gene from Pseudomonas aeruginosa that does confer robust resistance to 1) produces recombinant proteins that confer resistance to 1 and indicate that the FosA resistance proteins are functionally and genetically related to mlr3345.

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O2 to H2O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a “gate,” which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation.

Certain strains of the soil microorganism Streptomyces produce an antibiotic, fosfomycin [(1 R,2 S)-epoxypropylphosphonic acid], which is effective against both Gram-positive and Gram-negative pathogens by inhibiting the first committed step in cell-wall biosynthesis. Fosfomycin resistance proteins are metallo-enzymes that are known to inactivate the antibiotic by the addition of nucleophiles such as water, glutathione (GSH), l-cysteine and bacillithiol (BSH) to the oxirane ring of the molecule. Progress in the characterisation of FosB-type fosfomycin resistance proteins found in many Gram-positive organisms has been slow. This paper provides a brief description of the diversity of fosfomycin resistance proteins in general and, more specifically, new data characterising the substrate selectivity, structure, mechanism and metal-ion dependence of FosB enzymes from pathogenic strains of Staphylococcus and Bacillus. These new findings include the high-resolution X-ray diffraction structures of FosB enzymes from Staphylococcus aureus and Bacillus cereus in various liganded states and kinetic data that suggest that Mn(II) and BSH are the preferred divalent cation and thiol substrate for the reaction, respectively. The discovery of the inhibition of the enzyme by Zn(II) led to the determination of a ternary structure of the FosB·Zn(II)·fosfomycin·l-Cys complex which reveals both substrates present in a pose prior to reaction.