Eukaryotic-type serine/threonine kinase mediated phosphorylation at Thr 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 (mGmk) by eukaryotic-type Ser/Thr kinase, PknA. Mass spectrometric studies identified Thr and Thr 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 compared with Thr 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 might affect its interaction with Arg, 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, the interactions of Arg/Arg with Glu, Asp 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.