Phosphorylation of Enoyl-Acyl Carrier Protein Reductase InhA Impacts Mycobacterial Growth and Survival

Abstract

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...

Interaction of Mycobacterium tuberculosis Elongation Factor Tu with GTP Is Regulated by Phosphorylation

“…The kinase-dead mutant of PknB, PknB K40M , cloned into MCS2 along with MtbEf-Tu in MCS1, served as a control. The details of PknB cloning in pETDuet-1 have been described previously (25). The genes encoding PknB and PknB K40M were also cloned into pSD5 for overexpression in M. smegmatis as described previously (46).…”

“…The pathogenic bacterium Mycobacterium tuberculosis has 11 Ser/Thr protein kinases (STPKs), one Ser/Thr phosphatase (PstP), two Tyr phosphatases (16), and one Tyr kinase (9). PknB, an essential STPK of M. tuberculosis (42), has been associated with a large number of events relating to cell division and growth (18,23,25,31), metabolism (8,32,33), cell wall synthesis (20,51,52), and stress response (10,35). M. tuberculosis survives various stress conditions during disease establishment and has the ability to enter into dormancy.…”

“…Protein purification and biochemical assays. Proteins were expressed and purified from E. coli as described previously (8,20,25). In vitro phosphorylation of 5 g MtbEf-Tu/MtbEf-Tu T118A /Ef-Ts with PknB and/or other STPKs (0.5 to 3 g) was carried out in kinase buffer [20 mM piperazine-N,NЈ-bis(2-ethanesulfonic acid) (PIPES) (pH 7.2), 5 mM MnCl 2 , and 5 mM MgCl 2 for all the kinases and 25 mM Tris-Cl (pH 7), 1 mM dithiothreitol (DTT), 5 mM MgCl 2 , and 1 mM EDTA for PknE] containing 2Ci [␥-32 P]ATP (BRIT, Hyderabad, India), followed by incubation at 25°C for 20 min.…”

Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function

“…Bound proteins were eluted using 26 SDS sample buffer, and eluted proteins were precipitated with five volumes of cold acetone. Precipitated proteins were resuspended in 300 ml of 2D sample buffer and resolved on pH 3.5-5.6 IPG strips (GE) as described previously, followed by western blotting using anti-FLAG antibodies (Khan et al, 2010).…”

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.…”