Cloning,expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus

Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucoseconcentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning andcharacterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced(Accession number: {"type":"entrez-nucleotide","attrs":{"text":"JN645812","term_id":"374095279"}}JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clonewas purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weightof 33kDa. The rglk A showed very high affinity to glucose K_m 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis ofglucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially andphylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase(GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profoundrole in the pathogenesis.