Preliminary Purification And Enzymatic Characterization Of Pyridoxamine-pyruvate Aminotransferase From The Tobacco

Vitamin B₆（VB₆） is the general term for a kind of chemical compounds whichincluding the free forms of pyridoxine （PN）, pyridoxamine （PM）, pyridoxal （PL）, and thephosphate forms of pyridoxine-5’-phosphate （PNP）, pyridoxamine-5’-phosphate（PMP）,pyridoxal-5’-phosphate（PLP）. PLP acts as the essential coenzyme in manymetabolic conversions of amino acids. Tobacco as a model plant, also being used as theexperimental materials for the research of VB₆。It has reported that the determination ofVB₆vitamers in tobacco plants was PLP、PL and PN by using high performance liquidchromatography with fluorescence detector and it has been linked to stress responses inplants. Pyridoxamine-pyruvate aminotransferase catalyses the transfer of an amino groupbetween PM and pyruvate into PL and L-alanine,it was involved in a degradation pathwayfor VB₆.There were extensive researches of PPAT from the bacterias, but untile now therewas no report about the PPAT research from the plant. Tobacco is an important cash crop,in our experiment, we use the tobacco as the research object, extracted and investigatedenzymatic properties, establishes a foundation for study on the regulation mechanism ofPPAT in tobacco.The pyridoxamine–pyruvate aminotransferase was purified from the tobacco leaf byfreeze-drying, DEAE Sepharose Fast Flow ion exchange chromatography, SephadexG-100gel filtration and SP Sephadex C-25ion exchange chromatography. The enzymaticactivity and properties were investigated with phenyl hydrazine method. The resultsshowed that92.34-fold purification was obtained；The enzyme had an optimμm pH at9.0and it was stable at pH7.0⁹.0, It has an optimμm temperature at70℃and the enzyme hasgood thermal stability which remained about51.55%of its activity after being treated at80℃for3h; PLP and PMP did not enhance or reduce the enzyme activity,Hydroxylamine, phenylhydrazine and sodiμm borohydride, typical inhibitors ofPLP-dependent enzymes, did not inactivate the enzyme at all. Under optimal conditions,the Km values for pyridoxamine and pyruvate were6.337mmol/L and0.867mmol/L. Theresults provide a research basis for the metabolic mechanism of VB₆in tobacco plants.