Cloning, Expression, Purification and Characterization of UMP Kinase from Staphylococcus aureus

Autor: Abhijit Chaudhary, Potukuchi Venkata Gurunadha Krishna Sarma, O. Hari Prasad, O. V. S. Reddy, Y. Nanda Kumar
Rok vydání: 2012
Předmět:
Zdroj: The Protein Journal. 31:345-352
ISSN: 1875-8355
1572-3887
DOI: 10.1007/s10930-012-9410-0
Popis: Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150 kDa and exhibited monomeric form with molecular weight of 25 kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed Km of 2.80 ± 0.1 lM and Vmax 51.38 ± 1.39 l Mo f NADH/min/mg. The enzyme exhibited cooperative kinet- ics with ATP as substrate, as GTP decreased this cooper- ativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5a. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24 Aindicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials.
Databáze: OpenAIRE
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