title: Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis.
abstract: Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target.
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Nguyen-Hung
authors: NH
authors: Le
authors: the Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier, CNRS UMR 5235, Place Eugène Bataillon, 34095 Montpellier Cedex 05, and.
authors: Virginie
authors: V
authors: Molle
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Nathalie
authors: N
authors: Eynard
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Mathieu
authors: M
authors: Miras
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Alexandre
authors: A
authors: Stella
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Fabienne
authors: F
authors: Bardou
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Ségolène
authors: S
authors: Galandrin
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Valérie
authors: V
authors: Guillet
authors: the Institut Pasteur, Unité de Microbiologie Structurale, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors: Gwenaëlle
authors: G
authors: André-Leroux
authors: the Institut Pasteur, Unité de Microbiologie Structurale, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors: Marco
authors: M
authors: Bellinzoni
authors: the Institut Pasteur, Unité de Microbiologie Structurale, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors: Pedro
authors: P
authors: Alzari
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Lionel
authors: L
authors: Mourey
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS.
authors: Odile
authors: O
authors: Burlet-Schiltz
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, mamadou.daffe@ipbs.fr.
authors: Mamadou
authors: M
authors: Daffé
authors: From the Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, hedia.marrakchi@ipbs.fr.
authors: Hedia
authors: H
authors: Marrakchi
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D009171:Mycolic Acids / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009171:Mycolic Acids; C576758:polyketide synthase Pks13, Mycobacterium tuberculosis; D048630:Polyketide Synthases; D008025:Ligases
keywords: cell wall; lipid metabolism; mycobacteria; post-translational modification (PTM); protein phosphorylation