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

Abstract

Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential ␤-ketoacylacyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr 191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr 191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

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).…”

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

“…Signaling through STPKs has recently emerged as a key regulatory mechanism in M. tuberculosis, playing roles in the transport of metabolites (22), cell division (23), and virulence (24). Several metabolic pathways such as mycolic acid (25)(26)(27)(28), glutamine (29), phthiocerol dimycocerosates (30), and glucan (31) biosyntheses have been shown to be regulated by M. tuberculosis STPK phosphorylation.…”

The Staphylococcus aureus Autoinducer-2 Synthase LuxS Is Regulated by Ser/Thr Phosphorylation

“…To identify the number and nature of the phosphorylation sites, we opted for a mass spectrometry (MS) analysis, which was successfully developed previously (6,11,23,24). To this aim, LuxS was incubated with cold ATP in the presence of Stk1 and subjected to mass spectrometry analysis after tryptic digestion.…”

“…As revealed by the mapping on the LuxS structure, the Thr14 site appears critical for LuxS enzymatic activity. Therefore, following a strategy that has been successfully used to demonstrate that regulation of a substrate protein via phosphorylation is important during active cell growth in mycobacteria (Wag31) (12) or for mycolic acid metabolism (mtFabH or MabA) (23,24), we generated the phosphorylation mimic LuxS_T14D by site-directed mutagenesis (Tables 1 and 2) and expressed and purified it. Previous studies have shown that acidic residues such as Asp or Glu qualitatively recapitulate the effect of phosphorylation with regard to functional activity (12,23,24).…”