Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B6 metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5′-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr47, Ile54, Arg88, Asn121 and Glu230 might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn121 to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr47 to Asn, Ile54 to Phe, and Arg88 to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp230 to Glu. Circular dichroism analysis revealed that the mutation of Trp230 to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp230 is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK.

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B-6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 +/- 5.1 and 44.1 +/- 3.9 mu M. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight alpha-helices (alpha 1-8), nine beta-strands (beta 1-9) and two segments of 3(10) helices.