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

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 In...

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Autores principales: Vögeli, Bastian, Rosenthal, Raoul G., Stoffel, Gabriele M. M., Wagner, Tristan, Kiefer, Patrick, Cortina, Niña Socorro, Shima, Seigo, Erb, Tobias J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222099/
https://www.ncbi.nlm.nih.gov/pubmed/30217823
http://dx.doi.org/10.1074/jbc.RA118.005405
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author Vögeli, Bastian
Rosenthal, Raoul G.
Stoffel, Gabriele M. M.
Wagner, Tristan
Kiefer, Patrick
Cortina, Niña Socorro
Shima, Seigo
Erb, Tobias J.
author_facet Vögeli, Bastian
Rosenthal, Raoul G.
Stoffel, Gabriele M. M.
Wagner, Tristan
Kiefer, Patrick
Cortina, Niña Socorro
Shima, Seigo
Erb, Tobias J.
author_sort Vögeli, Bastian
collection PubMed
description 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.
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spelling pubmed-62220992018-11-08 InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis Vögeli, Bastian Rosenthal, Raoul G. Stoffel, Gabriele M. M. Wagner, Tristan Kiefer, Patrick Cortina, Niña Socorro Shima, Seigo Erb, Tobias J. J Biol Chem Enzymology 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. American Society for Biochemistry and Molecular Biology 2018-11-02 2018-09-14 /pmc/articles/PMC6222099/ /pubmed/30217823 http://dx.doi.org/10.1074/jbc.RA118.005405 Text en © 2018 Vögeli et al. Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle Enzymology
Vögeli, Bastian
Rosenthal, Raoul G.
Stoffel, Gabriele M. M.
Wagner, Tristan
Kiefer, Patrick
Cortina, Niña Socorro
Shima, Seigo
Erb, Tobias J.
InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis
title InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis
title_full InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis
title_fullStr InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis
title_full_unstemmed InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis
title_short InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis
title_sort inha, the enoyl-thioester reductase from mycobacterium tuberculosis forms a covalent adduct during catalysis
topic Enzymology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222099/
https://www.ncbi.nlm.nih.gov/pubmed/30217823
http://dx.doi.org/10.1074/jbc.RA118.005405
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