Cloning And Enzymology Analysis Of Aspartate Aminotransferase Gene From Tobacco

To maintain human health required a variety of vitamins,pyridine compounds Vitamin B6 was one of the indispensable,which had six forms,including free form pyridoxine(PN),pyridoxamine(PM),pyridoxal(PLP)and their corresponding phosphate forms Pyridoxine-5'-phosphate(PNP),Pyridoxamine-5'-phosphate(PMP)and pyridoxal phosphate(PLP).Among the six different forms PLP was the main form,participated in the catalytic response to amino acids as a coenzyme in many reactions.In the synthesis and metabolism process of many substances,PLP also played an important role.In nature,there were two metabolic pathways of vitamin B6--the de novo synthesis pathway and the salvage pathway.The salvage pathway involved the transformation between different forms of VB6 and also involved many enzymes such as pyridoxal reductase(PLR),pyridoxine oxidase(PNPO)Pyridoxal kinase(PLK)and phosphatase.Aspartate aminotransferase was an intracellular enzyme that existed in almost all eukaryotic organisms and was one of the most important transaminases in the organism.To date,it has been studied the relationship between aspartate aminotransferase and vitamin B6 metabolism,proved that the aspartate aminotransferase(GOT)could catalyze the transformation between PM and PL reaction after removing the coenzyme PLP in animals and microbes.Tobacco,as a model plant,was a good material for the study of plant physiology and biochemistry.In this study,the tobacco glutathione transaminase gene(named NtGOT)was obtained from NCBI online site.The gene was 1386 bp in length and encoded 462 amino acids.The molecular weight of the protein was 50.7KDa and the isoelectric point was 8.37.The amino acid ratio results showed that tobacco aspartate transaminase was highly conserved with many species.The pMal-c2x-NtGOT prokaryotic expression vector was constructed and successfully expressed in Escherichia coli.Though the enzyme activity reaction,we found that NtGOT could catalyze the transamination reaction between glutamic acid and oxaloacetate,and it was also involved in the conversion between PM and PL.What's more,coenzyme PLP had a great effect on the activity of NtGOT.In the experiment,we used the ammonium sulfate precipitation method to remove the coenzyme PLP from NtGOT and found that the rate of PM and PL reaction was gradually increased with the removement of PLP and the addition of PLP or PMP inhibited the conversion of PM and PL.It was also determined that the optimal amino receptor of NtGOT is oxaloacetate(OAA)in the conversion reactions.Under the optimum conditions we found that NtGOT protein's apparent Km = 2.35 mM,Vmax = 1.16 pmol pmol min-1 ug-1 protein.We had a conclusion that NtGOT did not participate in the transformation between PM and PL in tobacco as an enzyme directly,that its corresponding coenzyme binding site really worked.