4, 5 and 6 Thymidine kinase (TK) is the key enzyme in the pyrimidine salvage pathway, catalyzes the phosphorylation of thymidine–thymidine 5′-monophosphate (TMP).7 TK is important for cells engaged in active PD0325901 DNA synthesis and is regulated by feedback control mechanism mediated by thymidine 5′-triphosphate.8 Thus, formed dTMP is converted to dTDP by thymidine monophosphate kinase an enzyme which is junction between salvage and de novo biosynthesis. Therefore,

any variation in de novo or in salvage pathway the TMPK activity is very much influenced. TK and TMPK have been characterized in many bacteria and eukaryotes. 9, 10, 11 and 12 NMP kinases exhibit a protein fold featuring a central five-stranded β-sheet surrounded by helices.13 The protein can be divided into three parts, namely, the CORE region, the NMP-binding region, and the LID region. The CORE region is the most conserved among NMP kinases, comprising mainly β-sheets with surrounding α-helices, and contains the P-loop, which is the ATP binding site. The NMP-binding domain is largely helical among all NMP kinases except guanylate monophosphate kinases. The LID region covers part of the phosphate donor site. Substrate-induced conformational changes have been observed in various family members of NMP kinases with

large domain movements upon Vemurafenib supplier binding of one or both substrates.13 and 14 Distinct differences have been observed between human TMP kinases and bacterial TMP kinases and among various classes of bacteria.9, 10, 11 and 12 Moreover, human TK is present actively present only in the G phase of the cell whereas, TK is present in large amounts in S. Isotretinoin aureus and they normally by pass the ubiquitin mediated proteolysis 15 and therefore help the proliferation of S. aureus in the human host. Therefore, the present study is focused on the characterization of TK and TMPK genes of S. aureus, further its comparison with human TMPK and TK. S. aureus ATCC12600 was grown on modified Baird Parker media 16 and 17

at 37 °C. After overnight incubation single black shiny colored with distinct zone colony was picked and cultured in brain heart infusion (BHI) broth at 37 °C and this culture was used for the extractions of cytoplasm and chromosomal DNA. The cytosolic fraction was used for the TK and TMPK enzyme assay while chromosomal DNA is used for amplification of TK and TMPK genes. 16, 17 and 18 The enzyme activities were determined at 30 °C using coupled spectrophotometric assay on a Cyber lab spectrophotometer USA. One unit of TK activity is defined as the amount of enzyme catalyzing the production of 1 μmol nucleoside monophosphate per minute whereas one unit of TMPK activity is defined as the amount of enzyme catalyzing the production of 1 μmol nucleoside diphosphate per minute. The kinetic parameters Km and Vmax were evaluated from Hanes–Woolf plot ([S] vs [S/V]). Protein concentrations in all steps were determined by Bradford 1976 method.