title: The Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue.
abstract: Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C56-64-meromycolyl chain and a C24-26-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr45 as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a beta-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS UMR 5235, INSERM, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Veyron-Churlet
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Taylor
authors.forename: Alistair K
authors.initials: AK
authors.lastname: Brown
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zanella-Cléon
authors.forename: Klaus
authors.initials: K
authors.lastname: Fütterer
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009171:Mycolic Acids / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D009171:Mycolic Acids; C013596:meromycolic acids; D013912:Threonine; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C530127:FabH protein, Mycobacterium tuberculosis; D064429:Fatty Acid Synthases; C451020:PknA protein, Mycobacterium tuberculosis; C487814:PknF protein, Mycobacterium tuberculosis; D017346:Protein Serine-Threonine Kinases; D054889:Fatty Acid Synthase, Type II; C413881:fatty acid synthase I, mycobacteria