6EP8

InhA Y158F mutant in complex with NADH from Mycobacterium tuberculosis

Summary for 6EP8

• Entry DOI: 10.2210/pdb6ep8/pdb
• Descriptor: Enoyl-[acyl-carrier-protein] reductase [NADH], SODIUM ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (6 entities in total)
• Functional Keywords: mycolic acid biosynthetic process, oxidoreductase, therapeutic target, isoniazid, mutation, catalytic mechanism, enoyl thioester reductases, tuberculosis
• Biological source: Mycobacterium tuberculosis H37Rv
• Total number of polymer chains: 1
• Total formula weight: 30247.18

Authors

Wagner, T.,Voegeli, B.,Rosenthal, R.G.,Stoffel, G.,Shima, S.,Kiefer, P.,Cortina, N.,Erb, T.J. (deposition date: 2017-10-11, release date: 2018-09-19, Last modification date: 2024-01-17)

Primary citation

Vogeli, B.,Rosenthal, R.G.,Stoffel, G.M.M.,Wagner, T.,Kiefer, P.,Cortina, N.S.,Shima, S.,Erb, T.J.
InhA, the enoyl-thioester reductase fromMycobacterium tuberculosisforms a covalent adduct during catalysis.
J. Biol. Chem., 293:17200-17207, 2018

PubMed Abstract: The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of and is a key target of antituberculosis drugs to combat multidrug-resistant 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 ( the proton transfer), independently of the first half-reaction ( 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.

PubMed: 30217823
DOI: 10.1074/jbc.RA118.005405

Experimental method

X-RAY DIFFRACTION (1.8 Å)