The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, or MenD, catalyze the Stetter-like conjugate addition of α-ketoglutarate with isochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate and carbon dioxide. This reaction represents the first committed step for biosynthesis of menaquinone, or vitamin K2, a key cofactor for electron transport in bacteria and a metabolite for posttranslational modification of proteins in mammals. The medium-resolution structure of MenD from Escherichia coli (EcMenD) in complex with its cofactor and Mn2+ has been determined in two related hexagonal crystal forms. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes in which two of the domains bind and force the cofactor into a configuration that supports formation of a reactive ylide. The structures reveal a stable dimer-of-dimers association in agreement with gel filtration and analytical ultracentrifugation studies and confirm the classification of MenD in the pyruvate oxidase family of ThDP-dependent enzymes. The active site, created by contributions from a pair of subunits, is highly basic with a pronounced hydrophobic patch. These features, formed by highly conserved amino acids, match well to the chemical properties of the substrates. A model of the covalent intermediate formed after reaction with the first substrate α-ketoglutarate and with the second substrate isochorismate positioned to accept nucleophilic attack has been prepared. This, in addition to structural and sequence comparisons with putative MenD orthologues, provides insight into the specificity and reactivity of MenD and allows a two-stage reaction mechanism to be proposed.
crystal structure; enzyme mechanism; menaquinone biosynthesis; thiamine diphosphate cofactor
drug; enzyme; mechanism; tuberculosis
Staphylococcus aureus pathogenesis depends on a specialized protein secretion system, ESX-1, that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB; small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacteria tuberculosis suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and / or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins.
adaptor protein; chaperone; helical bundle; secretion system; virulence factor
A crystallographic and biochemical study of L. major cysteine synthase, which is a pyridoxyl phosphate-dependent enzyme, is reported. The structure was determined to 1.8 Å resolution and revealed that the cofactor has been lost and that a fragment of γ-poly-d-glutamic acid, a crystallization ingredient, was bound in the active site. The enzyme was inhibited by peptides.
Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K
i = 4 µM) by DYVI, a peptide based on the C-terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.
Arabidopsis thaliana; cysteine synthase; Leishmania major
4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp2 to sp3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism.
The structure of L. donovani pteridine reductase has been targeted to assist in a program of structure-based inhibitor research. Crystals that diffracted to 2.5 Å resolution were obtained and the structure has been solved. Unfortunately, the active site is disordered and this crystal form is unsuitable for use in characterizing enzyme–ligand interactions.
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.
antifolates; pteridine reductase; Leishmania; pterins; Trypanosoma
The first committed step in the classical biosynthetic route to menaquinone (vitamin K2) is a Stetter-like conjugate addition of α-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 Å) crystal structure of Bacillus subtilis MenD with cofactor and Mn2+ has been determined. Based on structure–sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of α-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure–reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.
CoA, coenzyme A; PDB, Protein Data Bank; SAD, single-wavelength anomalous diffraction; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate; SeMet, selenomethionine; ThDP, thiamine diphosphate; PEG, polyethylene glycol; crystal structure; enzyme mechanism; menaquinone biosynthesis; thiamine diphosphate cofactor
The 2.5 Å resolution structure of S. aureus adenylosuccinate lyase is reported and compared with those of orthologues to assess its potential as a template for early stage drug discovery. AMP and a putative assignment of oxalate, the latter an artefact possibly arising from an impurity in the PEG used for crystallization, occupy the active site.
The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit–subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization.
adenylosuccinate lyase; AMP; oxalate; purine biosynthesis; purine cycle
The close similarity of Trypanosoma brucei glutathione synthetase to the human orthologue indicates that the enzyme would be a difficult target for drug discovery.
Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-γ-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery.
AMP-PNP, adenylyl imidodiphosphate; GS, glutathione synthetase; GSH, glutathione; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid); MOPS, 3-(N-morpholino)-propanesulfonic acid; NCS, non-crystallographic symmetry; Tb, Trypanosoma brucei; TEV, tobacco etch virus; TLS, translation/libration/screw; TSA, trypanothione synthetase; T[SH]2, trypanothione; ATP-grasp; Glutathione; Glutathione synthetase; Trypanosoma brucei; Trypanothione; X-ray structure
The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.