title: Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase.
abstract: 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.
authors.affiliation: Key Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of China.
authors.forename: ShuoHao
authors.initials: S
authors.lastname: Huang
authors.forename: Ting
authors.initials: T
authors.lastname: Shu
authors.forename: JianYun
authors.initials: J
authors.lastname: Zhang
authors.forename: Wang
authors.initials: W
authors.lastname: Ma
authors.forename: Shu
authors.initials: S
authors.lastname: Wei
authors.forename: LongQuan
authors.initials: L
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011731:Pyridoxal Kinase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D019476:Insect Proteins; D011994:Recombinant Proteins; D011731:Pyridoxal Kinase