Enhancement of gene expression in Escherichia coli and characterization of highly stable ATP-dependent glucokinase from Pyrobaculum calidifontis

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_1032, annotated as glucokinase. Amino acid sequence analysis showed that Pcal_1032 belonged to ROK (repressor, open reading frame, and kinase) family of sugar kinases. To examine the properties of Pcal_1032, the coding gene was cloned and expressed in Escherichia coli. However, expression of the gene was low resulting in a poor yield of the recombinant protein. A single site directed mutation in Pcal_1032 gene, without altering the amino acid sequence, resulted in approximately tenfold higher expression. Purified recombinant Pcal_1032 efficiently phosphorylated various hexoses with a marked preference for glucose. ATP was the most preferred phosphoryl group donor. Optimum temperature and pH for the glucokinase activity of Pcal_1032 were 95 A degrees C and 8.5, respectively. Catalytic efficiency (k (cat)/K (m)) towards glucose was 437 mM(-1) s(-1). The recombinant enzyme was highly stable against temperature with a half-life of 25 min at 100 A degrees C. In addition, Pcal_1032 was highly stable in the presence of denaturants. There was no significant change in the CD spectra and enzyme activity of Pcal_1032 even after overnight incubation in the presence of 8 M urea. To the best of our knowledge, Pcal_1032 is the most active and highly stable glucokinase characterized to date from archaea, and this is the first description of the characterization of a glucokinase from genus Pyrobaculum.