InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis

Bastian Vögeli,Raoul G Rosenthal,Gabriele M.M Stoffel,Tristan Wagner,Patrick Kiefer,Niña Socorro Cortina,Seigo Shima,Tobias J Erb

doi:10.1074/jbc.ra118.005405

Abstract

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrug-resistant M. tuberculosis strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC–MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle. We used the isolated adduct as a molecular probe to directly access the second half-reaction of the catalytic cycle of InhA (i.e. the proton transfer), independently of the first half-reaction (i.e. the initial hydride transfer) and to assign functions to two conserved active-site residues, Tyr-158 and Thr-196. We found that Tyr-158 is required for the stereospecificity of protonation and that Thr-196 is partially involved in hydride transfer and protonation. The natural tendency of InhA to form a covalent C2-ene adduct calls for a careful reconsideration of the enzyme's reaction mechanism. It also provides the basis for the development of effective tools to study, manipulate, and inhibit the catalytic cycle of InhA and related enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily. In summary, our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs.

Highlights

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrugresistant M. tuberculosis strains
Our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs
Our data show that InhA forms a covalent adduct between the nicotinamide cofactor and the enoyl-CoA substrate during catalysis as observed in both pre–steady-state as well as steady-state (LC–MS) measurements

Summary

Results

Tyrosine 158 and threonine 196 are involved in catalysis Analysis of a crystal structure containing the cofactor NADϩ and a 16C-acyl-SNAC substrate analogue suggested a water molecule bound by threonine 196 that could be involved in catalysis and in proton donation (Fig. 1) [3]. This might be due to a water molecule in the active site able to compensate for the loss of the hydroxyl group of Tyr-158 as previously suggested by Parikh et al [7] Both variants T196A and T196V showed only a partial loss in stereospecificity, incorporating the deuteron into the 2R position with 91 Ϯ 1 and 77 Ϯ 1%, respectively (Table 2). The intramolecular Dkobs on protonation is determined by running assays in buffers with different H2O and D2O contents and fitting the isotopic composition of the products to Equation 1 [12] Using this direct method, we measured a Dkobs of 1.74 Ϯ 0.06 for InhA WT with hexenoyl-CoA as substrate. Detailed workup of the assay and analysis is described under “Experimental procedures.”

99 Ϯ 1 57 Ϯ 2 79 Ϯ 1 91 Ϯ 1 77 Ϯ 1

Discussion

Experimental procedures