Eukaryotic-type serine/threonine kinase mediated phosphorylation at Thr 169 perturbs mycobacterial guanylate kinase activity

S. Yadav, Ghanshyam and K. Ravala, Sandeep and Kachhap, Sangita and Thakur, Meghna and Roy, Abhishek and Singh, Balvinder and Karthikeyan, Subramanian and K. Chakraborti, Pradip (2017) Eukaryotic-type serine/threonine kinase mediated phosphorylation at Thr 169 perturbs mycobacterial guanylate kinase activity. Bioscience Reports, 37 (6). BSR20171048. ISSN 0144-8463

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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 Thr101 and Thr169 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 Thr169 compared with Thr101 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 Thr169 might affect its interaction with Arg166, 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 Thr169, the interactions of Arg165/Arg166 with Glu158, Asp121 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.

Item Type: Article
Additional Information: Copyright of this article belongs to Portland Press on behalf of the Biochemical Society
Uncontrolled Keywords: enzyme kinetics; mass spectrometry; molecular dynamic simulations; phosphorylation/dephosphorylation; site-directed mutagenesis
Subjects: Q Science > QR Microbiology
Depositing User: Dr. K.P.S.Sengar
Date Deposited: 28 Mar 2018 05:20
Last Modified: 28 Mar 2018 05:20

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