Human pyridoxal kinase: Overexpression and properties of the recombinant enzyme

Pyridoxal kinase catalyses the phosphorylation of the vitamin Bg. A human b rain pyridoxal kinase cDNA was isolated, and the recombinant enzyme was ove rexpressed in E. coli as a fusion protein with maltose binding protein (MBP ). Pure pyridoxal kinase exhibits a molecular mass of about 40 kDa when exa mined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a mono mer endowed with catalytic activity, indicating that the native quaternary structure of pyridoxal kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of pyrid oxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The K-m values for two substrates pyridoxal and ATP are 9 7 mu M and 12 mu M, respectively. In addition, the unfolding processes of t he recombinant enzyme were monitored by circular dichroism, The values of t he free energy change of unfolding (Delta G(o) = 1.2 kcal.mol(-1).K-1) and the midpoint transition (Ih I) suggested that the enzyme is more stable tha n ovine pyridoxal kinase against denaturation by guanidine hydrochloride. I ntrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residue s are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human pyridoxal kinase provide valuable informatio n for further biochemical studies on this enzyme.