Phosphorylation of enoyl-ACP reductase InhA impacts mycobacterial growth and survival

InhA, the primary target for the firstline anti-tuberculosis drug isoniazid, is a key enzyme of the Fatty Acid Synthase-II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In the present 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 Thr266 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 InhAT266E mutant, that mimics constitutive phosphorylation, is unable to rescue a M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr266 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.