title: Binding of NADP+triggers an open-to-closed transition in a mycobacterial FabG β-ketoacyl-ACP reductase.
abstract: The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining theMycobacterium smegmatisgenome uncovered several putative FabG-like proteins. Among them, we identifiedM. smegmatisMSMEG_6753 whose gene was found adjacent toMSMEG_6754, encoding a recently characterized enoyl-CoA dehydratase, and toMSMEG_6755, encoding another potential reductase. Recombinantly expressed and purified MSMEG_6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl-CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in afabGthermosensitiveEscherichia colimutant. Furthermore, comparison of theapoand the NADP+-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A ΔMSMEG_6753deletion mutant could be generated inM. smegmatis, indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.
authors.affiliation: Centre d'étude d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Université de Montpellier, CNRS, FRE 3689, 34293 Montpellier, France mickael.blaise@cpbs.cnrs.fr laurent.kremer@cpbs.cnrs.fr.
authors.forename: Mickaël
authors.initials: M
authors.lastname: Blaise
authors.affiliation: Centre d'étude d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Université de Montpellier, CNRS, FRE 3689, 34293 Montpellier, France.
authors.forename: Niël
authors.initials: N
authors.lastname: Van Wyk
authors.affiliation: Centre d'étude d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Université de Montpellier, CNRS, FRE 3689, 34293 Montpellier, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Banères-Roquet
authors.affiliation: Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 147, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq cedex, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Guérardel
authors.affiliation: Centre d'étude d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Université de Montpellier, CNRS, FRE 3689, 34293 Montpellier, France mickael.blaise@cpbs.cnrs.fr laurent.kremer@cpbs.cnrs.fr.|INSERM, CPBS, 1919 route de Mende, 34293 Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005816:Genetic Complementation Test; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D020102:Mycobacterium smegmatis / Q000737:chemistry* / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; C010667:acetoacetyl CoA; D009249:NADP; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D004746:Enoyl-CoA Hydratase
keywords: FabG; Mycobacterium; crystallography; short-chain dehydrogenase/reductase; β-ketoacyl reductase