Eukaryotic-type serine/threonine kinase mediated phosphorylation at Thr169 perturbs mycobacterial guanylate kinase activity

Guanylate kinase is an essential and conserved enzyme in nucleotide biosynthetic pathway that transfers phosphoryl group of ATP to GMP for yielding GDP. Here, we report the phosphorylation of guanylate kinase from Mycobacterium tuberculosis (mGmk) by eukaryotic-type Ser/Thr kinase, PknA. Mass spectrometric studies identified Thr 101 and Thr 169 as phosphorylatable residues in mGmk. To evaluate the significance of phosphorylation in these threonines, two point (T101A and T169A) and one double (T101A-T169A) mutants were generated. The kinase assay with these mutant proteins revealed the major contribution of Thr 169 compared with Thr 101 in the phosphorylation of mGmk. Kinetic analysis indicated that p-mGmk was deficient in its enzymatic activity compared with that of its un-phosphorylated counterpart. Surprisingly, its phosphoablated (T169A) as well as phosphomimic (T169E) variants exhibited decreased activity as was observed with p-mGmk. Structural analysis suggested that phosphorylation of Thr 169 might affect its interaction with Arg 166 , which is crucial for the functioning of mGmk. In fact, the R166A and R166K mutant proteins displayed a drastic decrease in enzymatic activity compared with that of the wild-type mGmk. Molecular dynamics (MD) studies of mGmk revealed that upon phosphorylation of Thr 169 , the interactions of Arg 165 /Arg 166 with Glu 158 , Asp 121 and residues of the loop in GMP-binding domain are perturbed. Taken together, our results illuminate the mechanistic insights into phosphorylation-mediated modulation of the catalytic activity of mGmk.