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

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 161; no. 2; pp. 155 - 160
• Main Authors: Huang, ShuoHao, Shu, Ting, Zhang, JianYun, Ma, Wang, Wei, Shu, Huang, LongQuan
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.02.2012
• Subjects: Amino Acid Sequence; Amino Acid Substitution; amino acids; Animals; Bombyx - enzymology; Bombyx mori; catalytic activity; Circular Dichroism; Cloning, Molecular; Conserved Sequence; enzymatic reactions; enzyme activity; Functional significance; Insect Proteins - biosynthesis; Insect Proteins - chemistry; Insect Proteins - genetics; Kinetics; mammals; metabolism; Molecular Sequence Data; mutagenesis; Mutagenesis, Site-Directed; mutants; phosphorylation; Protein Structure, Secondary; pyridoxal; Pyridoxal kinase; Pyridoxal Kinase - biosynthesis; Pyridoxal Kinase - chemistry; Pyridoxal Kinase - genetics; pyridoxine; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; sequence alignment; Site-directed mutagenesis; Bombyx mori; Functional significance; Pyridoxal kinase; Site-directed mutagenesis
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2011.10.009

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B 6 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 Thr 47, Ile 54, Arg 88, Asn 121 and Glu 230 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 Asn 121 to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr 47 to Asn, Ile 54 to Phe, and Arg 88 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 Trp 230 to Glu. Circular dichroism analysis revealed that the mutation of Trp 230 to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp 230 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.