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

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Vögeli,  Bastian
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Rosenthal,  Raoul
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Stoffel,  Gabriele
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Wagner,  Tristan
Department-Independent Research Group Microbial Protein Structure, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Cortina,  Nina Socorro
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Shima,  Seigo
Department-Independent Research Group Microbial Protein Structure, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Erb,  Tobias J.
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Citation

Vögeli, B., Rosenthal, R., Stoffel, G., Wagner, T., Kiefer, P., Cortina, N. S., et al. (2018). InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis. JOURNAL OF BIOLOGICAL CHEMISTRY, 293(44), 17200-17207. doi:10.1074/jbc.ra118.005405.


Cite as: http://hdl.handle.net/21.11116/0000-0004-4652-1
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.