The Mycobacterium tuberculosis β-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 C 56 -64 -meromycolyl chain and a C 24 -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 ␤-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 Thr 45 as the unique phosphoacceptor. This was further supported by complete loss of PknF-or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr 45 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 ␤-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.Within the infected host, Mycobacterium tuberculosis encounters numerous environmental conditions and induces or represses a number of genes for a quick adjustment to new conditions. The infection process of M. tuberculosis involves cross-talk of signals between the host and the bacterium, resulting in reprogramming of the host signaling network. Protein phosphorylation/dephosphorylation represent a central mechanism for distribution of signals to various parts of the cell, regulating growth, differentiation, mobility, and survival (1). In mycobacteria, a common signal transduction pathway is transmitted through membrane-embedded sensor kinases, enabling the pathogen to modify itself for survival in this hostile environment.Protein kinases can be classified into two families based on their similarities and enzymatic specification: (i) the histidine kinase superfamily, which relies on autophosphorylation of a conserved histidine residue ...

A Novel Mode of Regulation of the Staphylococcus aureus Catabolite Control Protein A (CcpA) Mediated by Stk1 Protein Phosphorylation

“…The MS/MS spectra unambiguously identified the presence of two phosphate groups on peptide(14 -30) ( Fig. 2A) and peptide (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42) (Fig. 2B), thus indicating that CcpA is phosphorylated on two threonines, corresponding to Thr-18 and Thr-33.…”

“…Lack of Complementation with the CcpA Phosphomimetic Mutant in ⌬ccpA Mutant Strain-Acidic residues such as aspartic acid (Asp) qualitatively mimic the phosphorylation effect with regard to functional activity (24,34,36,38,40,41). Therefore, phosphoablative (Thr to Ala replacements) and phosphomimetic (Thr to Asp replacements) ccpA alleles were generated and cloned into the E. coli-S. aureus shuttle plasmid pCN34 under the control of the ccpA promoter to trans-complement the S. aureus SA113 ⌬ccpA mutant KS66 (12).…”

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

“…The resulting construct was introduced by electroporation into M. bovis BCG using a Bio-Rad Gene Pulser. Transformants were grown and harvested at early stationary phase and the purification protocol was performed as previously described (32).…”

“…MabA Is Phosphorylated in Vitro on Three Threonine Residues-Mass spectrometry was used to identify the number and nature of the phosphorylation sites on MabA, a method that has been successfully used to elucidate the phosphoacceptors in a sequence-specific fashion for several other M. tuberculosis STPK substrates (32,39). Briefly, MabA was incubated with cold ATP in the presence of PknB (one of the most active kinases for MabA, Fig.…”

“…In our previous studies, the three condensing enzymes mtFabH, KasA, and KasB of the mycobacterial FAS-II system were identified as specific substrates of Ser/Thr protein kinases both in vitro and in vivo, and it was found that phosphorylation of these enzymes modulates their condensing activity (26,32). However, whether phosphorylation directly controls mycolic acid biosynthesis has not been clearly established.…”