Genevieve L. Evans

Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis.

The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 A (1 A=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (beta/alpha)(8)-barrel followed by a C-terminal alpha-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (beta/alpha)(8)-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr(143), Thr(54) and Tyr(117) appears to be disrupted. The kinetic parameters of the enzyme are reported: K(M)(ASA)=0.43+/-0.02 mM, K(M)(pyruvate)=0.17+/-0.01 mM and V(max)=4.42+/-0.08 micromol x s(-1) x mg(-1). Interestingly, the V(max) of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis

MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.

A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer-dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.1Å) revealed a dimeric protein with an identical fold and active-site structure to the tetrameric wild-type enzyme. Analytical ultracentrifugation confirmed the dimeric structure in solution, yet the dimeric mutant has similar activity to the wild-type enzyme. Although the affinity for both substrates was somewhat decreased, the high catalytic competency of the enzyme was surprising in the light of previous results showing that dimeric variants of the Escherichia coli and Bacillus anthracis DHDPS enzymes have dramatically reduced activity compared to their wild-type tetrameric counterparts. These results suggest that Mtb-DHDPS-A204R is similar to the natively dimeric enzyme from Staphylococcus aureus, and highlight our incomplete understanding of the role played by oligomerisation in relating protein structure and function.

Repurposing the Chemical Scaffold of the Anti‐Arthritic Drug Lobenzarit to Target Tryptophan Biosynthesis in Mycobacterium tuberculosis

The emergence of extensively drug‐resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need for new therapeutics to treat tuberculosis. We are attempting to fast‐track a targeted approach to drug design by generating analogues of a validated hit from molecular library screening that shares its chemical scaffold with a current therapeutic, the anti‐arthritic drug Lobenzarit (LBZ). Our target, anthranilate phosphoribosyltransferase (AnPRT), is an enzyme from the tryptophan biosynthetic pathway in Mtb. A bifurcated hydrogen bond was found to be a key feature of the LBZ‐like chemical scaffold and critical for enzyme inhibition. We have determined crystal structures of compounds in complex with the enzyme that indicate that the bifurcated hydrogen bond assists in orientating compounds in the correct conformation to interact with key residues in the substrate‐binding tunnel of Mtb‐AnPRT. Characterising the inhibitory potency of the hit and its analogues in different ways proved useful, due to the multiple substrates and substrate binding sites of this enzyme. Binding in a site other than the catalytic site was found to be associated with partial inhibition. An analogue, 2‐(2‐5‐methylcarboxyphenylamino)‐3‐methylbenzoic acid, that bound at the catalytic site and caused complete, rather than partial, inhibition of enzyme activity was found. Therefore, we designed and synthesised an extended version of the scaffold on the basis of this observation. The resultant compound, 2,6‐bis‐(2‐carboxyphenylamino)benzoate, is a 40‐fold more potent inhibitor of the enzyme than the original hit and provides direction for further structure‐based drug design.

Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

Datasets, processing and refinement details for Mtb-AnPRT: inhibitor structures with various space groups

There are twenty-five published structures of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase (Mtb-AnPRT) that use the same crystallization protocol. The structures include protein complexed with natural and alternative substrates, protein:inhibitor complexes, and variants with mutations of substrate-binding residues. Amongst these are varying space groups (i.e. P21, C2, P21212, P212121). This article outlines experimental details for 3 additional Mtb-AnPRT:inhibitor structures. For one protein:inhibitor complex, two datasets are presented – one generated by crystallization of protein in the presence of the inhibitor and another where a protein crystal was soaked with the inhibitor. Automatic and manual processing of these datasets indicated the same space group for both datasets and thus indicate that the space group differences between structures of Mtb-AnPRT:ligand complexes are not related to the method used to introduce the ligand.

A covalent adduct of MbtN, an acyl-ACP dehydrogenase from Mycobacterium tuberculosis, reveals an unusual acyl-binding pocket

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated with Mtb virulence. Here, the crystal structure of an Mtb enzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14-21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusual cis double bond in the mycobactin acyl chain.

Purification, crystallization and preliminary X-ray studies of MbtN (Rv1346) from Mycobacterium tuberculosis

In Mycobacterium tuberculosis, the protein MbtN (Rv1346) catalyzes the formation of a double bond in the fatty-acyl moiety of the siderophore mycobactin, which is used by this organism to acquire essential iron. MbtN is homologous to acyl-CoA dehydrogenases, whose general role is to catalyze the α,β-dehydrogenation of fatty-acyl-CoA conjugates. Mycobactins, however, contain a long unsaturated fatty-acid chain with an unusual cis double bond conjugated to the carbonyl group of the mycobactin core. To characterize the role of MbtN in the dehydrogenation of this fatty-acyl moiety, the enzyme has been expressed, purified and crystallized. The crystals diffracted to 2.3 Å resolution at a synchrotron source and were found to belong to the hexagonal space group H32, with unit-cell parameters a = b = 139.10, c = 253.09 Å, α = β = 90, γ = 120°.