Eukaryotic-type serine/threonine kinase mediated phosphorylation at Thr169 perturbs mycobacterial guanylate kinase activity
| Autor: | Ghanshyam S. Yadav, Balvinder Singh, Meghna Thakur, Subramanian Karthikeyan, Sandeep K. Ravala, Pradip K. Chakraborti, Sangita Kachhap, Abhishek Roy |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Serine/threonine-specific protein kinase biology MAP kinase kinase kinase Guanylate kinase Cyclin-dependent kinase 2 Biophysics Cell Biology Mitogen-activated protein kinase kinase Biochemistry MAP2K7 03 medical and health sciences 030104 developmental biology biology.protein Protein phosphorylation Cyclin-dependent kinase 9 Molecular Biology |
| Zdroj: | Bioscience Reports. 37 |
| ISSN: | 1573-4935 0144-8463 |
| DOI: | 10.1042/bsr20171048 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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