title: Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus.
abstract: Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization 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: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically 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 profound role in the pathogenesis.
authors.affiliation: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, India 517 507.
authors.forename: Hanumanthu Prasanna
authors.initials: HP
authors.lastname: Lakshmi
authors.forename: Sthanikam
authors.initials: S
authors.lastname: Yeswanth
authors.forename: Uppu Venkateswara
authors.initials: UV
authors.lastname: Prasad
authors.forename: Dudipeta
authors.initials: D
authors.lastname: Vasu
authors.forename: Vimjam
authors.initials: V
authors.lastname: Swarupa
authors.forename: Pasupuleti Santhosh
authors.initials: PS
authors.lastname: Kumar
authors.forename: Mangamoori Lakshmi
authors.initials: ML
authors.lastname: Narasu
authors.forename: Potukuchi Venkata Gurunadha
authors.initials: PV
authors.lastname: Krishna Sarma
keywords: Km; ROK; glk A; pQE 30