Site-directed Mutation Of Pyridoxine 5'-phosphate Oxidase From Bombyx Mori In Vitro And Expression, Bio-activity Assay Of The Mutants

Vitamin B6 is a group of water-soluble vitamin, and also is the general term for a class of pyridine compounds,2-methyl-3-hydroxy-5-hydroxymethylpyridine is their common matrix. The free forms are pyridoxine(PN), pyridoxamine(PM), pyridoxal(PL) and the phosphate forms are pyridoxine-5'-phosphate(PNP), pyridoxamine-5'-phosphate(PMP), pyridoxal-5'-phosphate(PLP). PLP is one of the major coenzyme form of VB6. PLP is an important coenzyme in a variety of amino acid metabolism of enzymes, it involves in all kinds of amino acid catalytic, plays an important role in the synthesis of amino acids, decompose and conversions.The biosynthesis method of PLP includes two way, de novo and salvage pathway. Pyridoxine-5'-phosphate oxidase(PNPO) playes a key role in salvage pathway. In the presence of flavin mononuleotide(FMN) and O2, PNPO oxidizes the alcohol or amino on PNP and PMP to generate for PLP.Bombyx mori(B.mori) is the major model of the study of lepidoptera genomes and genes, and is second only to drosophila as a representative of insect genes. The study of functional gene of the silkworm not only helps clarify the function of homologous genes, but also pushes the process such as the insect domestication, morphogenesis, endocrinology, reproduction, behavior and immunity. In the silkworm silk protein synthesis and the process of growth and development, VB6 metabolism is an important part. PNPO is the only flavoprotein oxidase that can oxidizes amino or alcohol-based to aldehyde. It may be a critical control point in VB6 metabolic process.The results showed that the site-directed mutagenesis of Lyslll and Ser160 of Bombyx mori PNPO cDNA respectively. Lysine mutated to the glutamic acid, which marked as K-E, Serine mutated to the alanine, which marked as S-A. Identification of the mutant site functions makes use of recombinant expression and in vitro activity determination.The protein molecular weight of mutant recombinant silkworm PNPO detected by SDS-PAGE is about 45.0 kDa, was consistent with the former. In determination of in vitro enzymatic reactions,K-E mutant activity decreased by about 78.0%; S-A mutant enzyme activity decreased by about 67.4%. This study made clear the significance of some conserved amino acid residues on the catalytic function of PNPO of Bombyx mori.