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InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.The characteristic nature of the cell envelope of Mycobacterium tuberculosis is linked to its pathogenicity. The thick layer of lipids on the outer surface of mycobacteria is protective in nature, and mycolic acids comprise the bulk of this layer. Mycolic acids also constitute structural components of the cell wall and envelope (1, 2). Mycobacteria are unique in having two fatty-acid synthase (FAS) 5 systems, FAS-I and FAS-II, and both of these pathways are involved in the synthesis of mycolic acids. Eukaryotic-like FAS-I is a single multidomain enzyme, whereas FAS-II includes discrete monofunctional enzymes that carry out the various sequential steps involved in the process of synthesis (3). The FAS-I system is responsible for the de novo fatty acid biosynthesis (4), producing C20-and C26-S-enzyme derivatives that are converted to the respective coenzyme A (CoA) forms and then released. The C20 fatty acid is the probable starting point for the FAS-II system (2), and the C26 fatty acid is incorporated as the ␣-branch of mycolic acid (5).FabH (␤-ketoacyl-ACP synthase III) forms the link between the two FAS systems by condensing the long chain acyl-CoA products (produced by FAS-I) and malonyl-ACP (produced from malonyl-CoA by the action of FabD (malonyl-CoA:ACP transacylase) (6)). The ␤-ketoacyl-S-ACP product (7) thus formed is subjected to keto-reduction, dehydration, and en...

The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ⑀-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (⑀-amine) and Thr (␣-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.

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