Research Summary – Department of Chemistry, Stony Brook University, Stony Brook, NY

InhA catalyzes the NADH-dependent reduction of 2-trans-enoyl-ACP’s and is involved in the biosynthesis of mycolic acid, a critical component of the mycobacterial cell wall. This enzyme is the target for the anti-tubercular drugs isoniazid and ethionamide and mutations in the enzyme result in drug-resistant strains of M. tuberculosis.

Mechanism of Isoniazid Action

Isoniazid (INH) is one of the most effective and commonly prescribed anti-tubercular drugs. Isoniazid is a pro-drug that is first activated by the mycobacterial catalase-peroxidase enzyme, KatG. The activated form of isoniazid becomes covalently attached to the nicotinamide ring of the NADH bound within the active site of InhA, creating an INH-NAD metabolite that acts as a tight binding inhibitor. More importantly, mutations in InhA and KatG lead to resistance to isoniazid. Currently five InhA mutations (I16T, I21V, I47T, V78A, and I95P) have been observed in clinical isolates of INH-resistant M. tuberculosis. All the mutations are in or adjacent to the NADH binding pocket and result in reduced affinity for NADH while leaving the kcat and Km for the enoyl substrate unaffected. We have determined the rate of inactivation of these INH-resistant mutants with activated isoniazid.

Role of Tyrosine 158 and Lysine 165 in the Catalytic Mechanism of InhA

We have investigated the role of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA by site-directed mutagenesis, equilibrium binding, steady state enzyme kinetics and kinetic isotope effects. These two residues have been identified as putative catalytic residues on the basis of structural and sequence homology with short chain alcohol dehydrogenase (SCAD) family of enzymes. Replacement of Y158 with phenylalanine (Y158F) and alanine (Y158A) results in a 24-fold and 1,500-fold decrease in kcat, respectively, while leaving the Km for the substrate unaffected. Replacement of Y158 with serine (Y158S) results in an enzyme with wild-type activity. Replacement of K165 with glutamine (K165Q) and arginine (K165R) has no effect on the enzymes’ catalytic ability or its ability to bind NADH. However, substitution of alanine or methionine for lysine at this position results in reduced affinity for NADH. The data here suggests that tyrosine 158 is involved in catalysis while the primary role of lysine 165 is in cofactor binding.

Mechanism of InhA Inhibition by Triclosan

Recently it was reported that the ‘nonspecific’ antibacterial compound triclosan is a specific inhibitor of EnvM, the enoyl-ACP reductase from E. coli.   The X-ray structure of triclosan bound to EnvM at 1.75Å.  Since InhA and EnvM have 46% sequence identity and share a high degree of 3D structural homology, it is thought that triclosan interacts with both enzymes in a similar fashion.  We have shown that triclosan is a submicromolar inhibitor of wild-type InhA.  Binding of triclosan to wild-type protein in uncompetitive with respect to NADH and 2-trans-dodecenoyl-CoA, giving K_i’ values of 0.22 ± 0.02 and 0.21 ± 0.01 mM, respectively.  Based on the crystal structure of triclosan bound to EnvM, we hypothesized that the conserved tyrosine residue in InhA (Y158) might interact with triclosan. Replacing Y158 with phenylalanine (Y158F) reduces the affinity for triclosan over 200-fold.  Levy and coworkers identified four InhA mutations (M161V, M103T, A124V and S94A) that resulted in resistance of M. smegmatis to triclosan.  We have generated these mutations in the M. tuberculosis protein and studied their inhibition by triclosan.