Influences Of Mutation At Acetylated Sites On The Catalytic Activity Of Escherichia Coli Pyridoxine5’-Phosphate Oxidase

Escherichia coli pyridoxine5’-phosphate oxidase catalyzes the oxidation of either pyridoxine5’-phosphate or pyridoxamine5’-phosphate to form pyridoxal5’-phosphate. This reaction served as the terminal step in the de novo biosynthesis of pyridoxal5’-phosphate in E. coli and as a part of the salvage pathway in both E. coli and mammalian cells. Acetylation was broadly found in many metabolic enzymes. Recently, acetylation was reported to be related to cellular dynamics, apoptosis, energy metabolism, protein transportation and autophagy.The results of proteome microarrays constructed by our laboratory showed that E. coli pyridoxine5’-phosphate oxidase might involve in acetylation modification. Our research determined the acetylation modification of E. coli pyridoxine5’-phosphate oxidase by Western blotting and identified two acetylated lysine residues(72Lys and159Lys) by mass spectrometry. Multiple sequence alignment of pyridoxine5’-phosphate oxidase from different organisms indicated that72Lys was highly conserved and159Lys showed lower conservation. Site-direct mutants of acetylated sites were constructed and72Lys was proved to be very critical for keeping the catalytic activity of E. coli pyridoxine5’-phosphate oxidase. Except for K72R, the other three mutants(K72Q, K72P and K72A) exhibited less than10%catalytic activity and showed different kinetic constants from the wild type. Moreover, mutation for72Lys changed the optimal pH, temperature of the enzymatic reaction and the thermal stability of the oxidase; while, mutants of159Lys showed almost equal catalytic activity to the wild type.