Abstract
Aim: To characterize, TK1111, a cupin-type phosphoglucose isomerase from a hyperthermophilic archaeon Thermococcus kodakarensis. Methods: The genome of T. kodakarensis was searched in order to find an open reading frame annotated as glucose-6-phosphate isomerase. The structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Effect of metal ions on the restoration of enzyme activity was examined. Results: Genome search of T. kodakarensis revealed the presence of an open reading frame, TK1111, annotated as glucose-6-phosphate isomerase. TK1111 exhibited high homology with archaeal PGIs related to the cupin-superfamily instead of well-known family of PGI enzymes. The gene encoding TK1111 consisted of 567 nucleotides corresponding to a polypeptide of 189 amino acids. The structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Recombinant TK1111 displayed highest activity (43 U/mg) at 90 °C and pH 6.0. To our knowledge this is the only PGI from cupin-superfamily exhibiting highest activity in the acidic pH. TK1111 catalyzed the conversion of fructose 6-phosphate to glucose 6-phosphate following Michaelis-Menten kinetics with a Km and Vmax values of 2.2 mM and 12.5 mmol min -1 mg-1, respectively at 37 °C. Addition of Zn2+ ions in the assay mixture remarkably enhanced the enzyme activity in contrast to the PGI from Thermococcus litoralis, another member of the same genus exhibiting 75% identity to TK1111. The enzyme activity was completely diminished when TK1111 was heated at 70 °C in the presence of EDTA. Complete restoration of the enzyme activity was observed when TK1111 was incubated at room temperature in the presence of Zn2+. Conclusion: TK1111 from T. kodakaraensis is a novel phosphoglucose isomerase that belongs to cupintype isomerases. To our knowledge this is the only PGI from cupin-family exhibiting highest activity in the acidic pH. Enzyme activity was remarkably enhanced in the presence of Zn2+ in contrast to other members of the family.
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