Phosphorylation of enoyl-acyl carrier protein reductase InhA impacts mycobacterial growth and survival.
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.
Khan
Shazia
S
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067 India.
Nagarajan
Sathya Narayanan
SN
Parikh
Amit
A
Samantaray
Sharmishtha
S
Singh
Albel
A
Kumar
Devanand
D
Roy
Rajendra P
RP
Bhatt
Apoorva
A
Nandicoori
Vinay Kumar
VK
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000254:growth & development* / Q000502:physiology; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D017346:Protein Serine-Threonine Kinases