CDNA Cloning, Expression In E.coli, Enzymatic Identification And Genomic Organization Of Two Key Enzymes, Which Are Involved In The VB6 Metabolism Of Bombyx Mori

Vitamin B6(VB6)is the collective term for a group of compounds,pyridoxine(PN), pyridoxal(PL)and pyridoxamine(PM),and their phosphorylated derivatives,pyridoxine 5'-phosphate(PNP),pyridoxal 5'-phosphate(PLP)and pyridoxamine 5'-phosphate(PMP). PLP is a cofactor that is required by more than 100 cellular enzymes,the majority of which are involved in amino acid metabolism.PLP is also involved in the other metabolic path-ways,and play an important role in cells.In plants and many microorganisms,PLP is syn-thesized from small metabolites by a de novo pathway.There are two de novo pathways of PLP biosynthesis in nature.One is related to PdxA and PdxJ,the other is related to PDX1 and PDX2.Mammals do not have the biosynthetic pathway and require the precursors of VB6,that is,PN,PL,or PM,from their diet.In fact,both bacteria and eukaryotic cells have a salvage pathway for reutilizing the PLP during protein turnover.The vitamin B6 salvage pathway is involved in interconversions between different B6 vitamers.Pyridoxal kinase(PLK),Pyridoxine(pyridoxamine)5'-phosphate oxidase(PNPO),and vitamin B6 phosphatase are all involved in the salvage pathway.In the presence of Zn²⁺and ATP,PLK catalyzes the phosphorylation of PN,PL,and PM to form PNP,PLP,PMP.PNPO catalyzes the oxidation of PNP and PMP to form PLP,in the presence of FMN and O₂.Bombyx mori cannot synthesize PLP from small metabolites by de novo pathway and, similar to mammals,relies on a nutritional source of VB6 to synthesize PLP.However, there are some differences between Bombyx mori and mammals in the VB6 metabolism. In point of immense protein turnover in Bombyx mori,the homeostasis of PLP concentra-tion is to be maintained more intensively.Therefore,the activity regulation of PLK and PNPO is more obvious in Bombyx mori.The cDNAs or genes of PLK and PNPO have been identified from mammals,plants, and several microorganisms.However,no study of PLK or PNPO gene cloning was re-ported in an insect.Some structural and kinetic studies have been done on the two enzymes from several species,although their catalyzing mechanisms are remained to be further studied.Based on the genomic and EST database of Bombyx mori,the putative cDNAs encod-ing PLK and PNPO were obtained using the method of bioinformatics.The primers that encompassed the ORF of putative PLK-gene and PNPO-gene were designed according to the putative cDNAs.By RT-PCR using those primers and the total cDNA of Bombyx mori fat body as templates,two PCR products with expected molecular size were generated.The PCR products were cloned and sequenced,and the sequences were accepted by GenBank under the accession number of DQ452397 and DQ452398.The PLK-cDNA has a 894-bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.09 kDa.The amino acid sequence shares 52.84%identity with human PLK,and it also contains signature conserved motifs of PLK family.However,the protein is 10 aa shorter than PLK from mammals and plants.The PNPO-cDNA has a 771-bp open reading frame and encodes a protein of 257 amino acid residues with a molecular mass of 29.86 kDa.The amino acid sequence shares 51.44%identity with human PNPO,and some con-served amino acid residues also exist in it.Some conserved amino acid residues in human and E.coli PNPO,which play important roles in the enzyme,are changed in Bombyx mori.The ORF of the two cDNAs were cloned into pET22b(+)to construct expressed vec-tors.The expressed vectors were transformed to E.coli BL21(DE3),and expected non-fused proteins were expressed efficiently after strains were induced and cultured.The en-zymatic activity of crude extracts containing expressed products were measured by a fluorometric assay.The PNPO enzymatic activity was measured using the two enzymes of PLK and PNPO in the first.The crude extracts containing the expressed product of PLK-cDNA have strong enzymatic activity of pyridoxal kinase with a value of 30 nmol/min/mg. The crude extracts containing the expressed product of PNPO-cDNA have strong enzy-matic activity of pyridoxine 5'-phosphate oxidase with a value of 17 nmol/min/mg.The re-sults of enzymatic assay confirmed that the two cDNAs are authentic cDNAs encoding PLK and PNPO of Bombyx mori.According to genomic database of Bombyx mori and the cDNAs of PLK and PNPO, the gene organization of PLK and PNPO were determined.The Bombyx mori PLK gene is composed of five exons and four introns,and spans approximately 10 kb.The Bombyx mori PNPO gene is also composed of five exons and four introns,and spans approximately 3.5 kb.All exon/intron junctions of PLK and PNPO gene of Bombyx mori contain the gt/ag consensus splicing site.The TATA-like box and CAAT-like box are found in both of regulatory regions of PLK and PNPO gene.