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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...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2018
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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. |
format | Online Article Text |
id | pubmed-6222099 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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