Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation
| Autor: | P. Santhosh Kumar, D. Kavya, Uppu Venkateswara Prasad, Lokanathan Srikanth, Abhijit Chaudhury, Potukuchi Venkata Gurunadha Krishna Sarma, Y. Nanda Kumar, Vimjam Swarupa, C.H. Lalith Kumar, G. Sowjenya, Sthanikam Yeswanth, Kumar Venkatesh, D. Vasu |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
chemistry.chemical_classification
Staphylococcus aureus business.industry Process Chemistry and Technology Biofilm Inosine monophosphate dehydrogenase medicine.disease_cause Molecular biology Michaelis–Menten kinetics Microbiology Enzyme Fuel Technology chemistry IMP dehydrogenase Medicine Economic Geology Enzyme kinetics business Purine metabolism Escherichia coli Polyacrylamide gel electrophoresis |
| Zdroj: | Journal of Clinical and Scientific Research, Vol 2, Iss 4, Pp 203-210 (2013) |
| ISSN: | 2277-8357 2277-5706 |
| Popis: | Background: In Staphylococcus aureus purine metabolism plays a crucial role in the formation of biofilm which is a key pathogenic factor. The present study is aimed in the characterization of inosine monophosphate dehydrogenase (IMPDH) from Staphylococcus aureus ATCC 12600. Methods: IMPDH gene was amplified using primers designed from IMPDH gene sequence of S. aureus reported in the database. Then polymerase chain reaction (PCR) product was cloned in the Sma I site of M13mp18 and expressed in Escherichia coli JM109. The recombinant IMPDH (rIMPDH) was overexpressed with 1 mM isopropyl beta-D-1- thiogalactopyranoside (IPTG); Michaelis constant (Km), maximum enzyme velocity (Vmax) and catalytic constant (Kcat) of expressed IMPDH were determined. Results: The enzyme kinetics of IMPDH grown under aerobic conditions showed a Km of 43.71±1.56 µM, Vmax of 0.247±0.84/µM/mg/min and Kcat of 2.74±0.015/min while in anaerobic conditions the kinetics showed Km of 42.81±3.154/ µM, Vmax of 0.378±0.036 µM/mg/min and Kcat of 4.78±0.021 /min, indicating elevated levels of IMPDH activity under anaerobic conditions. Three-folds increased activity in the presence of 1 mM adenosine triphosphate (ATP) correlated with biofilm formation. The kinetics of pure rIMPDH were close to the native IMPDH of S. aureus ATCC12600 and the enzyme showed single band in sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of 53 KDa. Conclusions: Elevated activity of IMPDH was observed in S. aureus grown under anaerobic conditions and this was correlated with the biofilm formation indicating the linkage between purine metabolism and pathogenesis. |
| Databáze: | OpenAIRE |
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