Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase

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.