Cloning And Enzymatic Analysis Of Pyridoxal-5'-phosphate Phosphate Gene From Glycine Max

Vitamin B6?VB6?was a general term for six pyridine derivatives that could be converted to each other.It was the coenzyme for a variety of enzyme,played an important role in many metabolic reactions.Pyridoxal-5'-phosphateate?PLP?was the main form of vitamin B6 as coenzyme.PLP participated in amino acid metabolic transformations in the form of coenzymes.There was a active aldehyde in PLP's chemical structure.In the body,free PLP combined with amine-containing compounds and producted the formation of aldehydes and other substances in the role of active aldehyde.What's more,this binding reaction was likely to occur,which may affect the normal physiological level of amine group or other substance in a living body.The dynamic balance of PLP played an important role in maintaining the normal physiological function of mammals.PLP metabolism research in vivo was more in mammals than in plants,but how to maintain a dynamic balance mechanism was not clear.PLP levels in the body were regulated by factors such as PLP phosphatase,PL kinase and PNP oxidase.The concentration of free PLP in vivo required a certain mechanism to regulate,and PLP hydrolysis is an important regulatory mechanism.Studies on the cloning and analysis of PLP-related enzymes were the basis for clarifying the mechanism of PLP hydrolysis.In this study,soybean was the experimental material for the soybean seeds were rich in plant protein,and amino acid metabolism was very active in the corresponding physiological process,so maintain the dynamic balance of PLP concentration in vivo was very important for soybean.In this experiment,soybean was used as the experimental material.The target gene Soybean Phosphoric Acid Pyridoxal Phosphatase?GmPLPP??Gene ID:100806718?which has been named but not verified was found in the NCBI online website and the primers were designed to clone GmPLPP.Prokaryotic expression was carried out in the expressed strain to obtain the target protein.The objective protein was purified and the related enzymatic properties were studied.At the same time,the RNAi vector of GmPLPP was constructed.The cDNA of soybean GmPLPP gene cloned in this study is 948bp long and encoded315 amino acids with a corresponding molecular weight of about 36.91 kDa.Nucleotide sequence analysis showed that the consistency of GmPLPP with human and mouse specific PLP phosphatase was 36%.Amino acid sequence analysis showed that the amino acid sequence of GmPLPP and the amino acid sequence of human and mouse specific PLP phosphatase were relatively conserved at the amino acid sites associated with the active site,and they contained multiple homologous protein translational modification sites?Including 1 N-glycosylation site,3 phosphorylation sites,and 3 N-myristoylation sites?.The PLPP gene predicted by soybean was compared with the PLPP gene predicted in human,mouse and other predicted PLPP gene.The results showed that there was a conserved amino acid-aspartate in the PLPP gene of multiple species.And it was found that the amino acid sequence of PLPP in animals was highly consistent,and the PLPP amino acid sequence in plant was also highly consistent,but the PLPP amino acid sequence between animals and plants was not uniform,which might be related to the species differences.The results showed that the optimum pH of the target enzyme was about 7.5 and the optimum temperature was 45?.Mg²⁺had a catalytic effect on the enzymatic reaction of the enzyme and could release the inhibitory effect of low concentration of EDTA on the enzymatic reaction.The Ca²⁺,Zn²⁺had a weak inhibitory effect on the enzymatic reaction;Mn²⁺and Cu²⁺had a strong inhibitory effect on the enzymatic reaction,this effect increased with the increasing of the metal ionconcentration.Under the optimum enzymatic reaction conditions,the Km values of p-nitrophenyl phosphate?pNPP?,pyridoxal phosphate?PLP?and pyridoxamine phosphate?PMP?as substances were 0.608 mmol/L,0.45 mol/L and 0.637mol/L,Vmax were 0.703umol/min/?g protein,0.814 umol/min/?g protein and 0.967 umol/min/?g protein.Since the Km of PLP was smaller than that of pNPP and PMP,it indicated that the affinity of PLP was relatively high.The enzyme activity assay using a variety of phosphate-type compounds as substrates showed that the enzyme had some activity,but had the largest activity for PLP.According to the effect of different compounds on the enzymatic reaction of PLP as substrate,it was found that imidazole had a competitive inhibitory effect on the enzymatic reaction.Furthermore,pNPP,PMP,Na₃PO₄,Na₂MoO₄,Na₄P₂O₇ and KF inhibited the enzymatic reaction and the inhibition increased with its increasing concentration.This study had successfully constructed the RNAi vector of GmPLPP for subsequent research.