Molecular cloning, expression in Escherichia coli and structural-functional analysis of a pyruvate kinase from...

This manuscript describes recombinant production, characterization and structural analysis of wild-type and mutant Pcal_0029, a pyruvate kinase from Pyrobaculum calidifontis. Recombinant Pcal_0029 was produced in soluble and highly active form in Escherichia coli. Purified protein exhibited divalent metal-dependent activity which increased with the increase in temperature till 85 °C. Recombinant Pcal_0029 was highly thermostable with no significant loss in activity even after an incubation of 120 min at 100 °C. The enzyme exhibited apparent S_0.5 and V_max values of 0.44 ± 0.05 mM and 840 ± 39 units, respectively, towards phosphoenolpyruvate. These values towards adenosine-5'-diphosphate were 0.5 ± 0.07 mM and 870 ± 26 units, respectively. In silico structural analysis and comparison with the characterized enzymes revealed the presence of eight conserved regions. Two substitutions, K130E and S155G, resulted in a 10-fold decrease in activity. Secondary structure analysis indicated similar structures for the wild-type and the mutant enzymes. Bioinformatics analysis revealed disruption of interatomic interactions and hydrogen bonds, leading to a decreased flexibility and solvent accessibility, which may have led to decrease in activity. To the best of our knowledge, Pcal_0029 is the most thermostable pyruvate kinase reported so far. Moreover, this is the first study on the role of non-catalytic residues in a pyruvate kinase.