K-090. Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-ACP Synthase III (FabH) by Serine/Threonine Protein Kinases

R. Veyron-Churlet1, V. Molle2, L. Kremer1;
1CNRS-Univ. de Montpellier II, Montpellier, FRANCE, 2CNRS-Univ. de Lyon I (IBCP), Lyon, FRANCE.

Mycolic acids are key components of the Mycobacterium tuberculosis cell wall. These α-alkylated β-hydroxylated fatty acids are formed by the condensation of two fatty acids, a long C56 meromycolyl chain and a shorter C26 fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS) systems with FAS-I products being elongated by FAS-II towards meromycolic acids. The β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. The first step of the reaction catalyzed by FabH transfers an acyl group from acyl-CoA to the active site of the enzyme, followed by a two-carbon acyl chain extension through Claisen-type condensation with malonyl-ACP. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism because of its involvement in virulence. M. tuberculosis has eleven STPKs but the nature and function of their substrates remain largely unknown. Here, we provide evidence that FabH is a substrate of M. tuberculosis STPKs. In vitro kinase assays using [γ-33P]ATP demonstrated that FabH could be phosphorylated by different kinases, in particular by PknF. Analysis of the phosphoamino acid content indicated that FabH was phosphorylated exclusively on threonine residues by PknF. MALDI-TOF was used in combination with LC-ESI/MS/MS to identify and localize the phosphorylation site(s). Thr45 was found to be the only phosphorylated residue. Moreover, substitution of Thr45 by Ala completely abrogated PknF-dependent phosphorylation of FabH. Studies are currently in progress to determine how phosphorylation of Thr45 modifies the 3D structure of the condensing enzyme, whether it modulates the condensing activity and affects mycolic acid biosynthesis. Phosphorylation of FabH may represent a post-translational modification, which could participate in mycolic acid regulation, particularly required for adaptation and intracellular survival of pathogenic mycobacteria in host macrophages.