Structural and functional investigations of Pcal_0606, a bifunctional phosphoglucose/phosphomannose isomerase from Pyrobaculum calidifontis

We are investigating the glycolytic pathway in Pyrobaculum calidifontis whose genome sequence contains homologues of all the enzymes involved in this pathway. We have characterized most of them. An open reading frame, Pcal_0606, annotated as a putative phosphoglucose/phosphomannose isomerase has to be characterized yet. In silico analysis indicated the presence of more than one substrate binding pockets at the dimeric interface of Pcal_0606. The gene encoding Pcal_0606 was cloned and expressed in Escherichia coli. Recombinant Pcal_0606, produced in soluble form, exhibited highest enzyme activity at 90 °C and pH 8.5. Presence or absence of metal ions or EDTA did not significantly affect the enzyme activity. Under optimal conditions, Pcal_0606 displayed apparent Km values of 0.33, 0.34, and 0.29 mM against glucose 6-phosphate, mannose 6-phosphate and fructose 6-phosphate, respectively. In the same order, Vmax values against these substrates were 290, 235, and 240 μmol min−1 mg−1, indicating that Pcal_0606 catalyzed the reversible isomerization of these substrates with nearly same catalytic efficiency. These results characterize Pcal_0606 a bifunctional phosphoglucose/phosphomannose isomerase, which displayed high thermostability with a half-life of ∼50 min at 100 °C. To the best of our knowledge, Pcal_0606 is the most active and thermostable bifunctional phosphoglucose/phosphomannose isomerase characterized to date.