Study On Biochemical Characteristics And Catalytic Mechanism Of N-acetylornithine Aminotransferase From Synechocystis Sp. PCC6803

N-acetylornithine aminotransferase is a key enzyme in the arginine synthesis pathway which catalyzes the conversion of N-acetylornithine to N-acetylglutamate semialdehyde by using PLP as cofactor.N-acetylornithine aminotransferase from Synechocystis sp.PCC6803 has the function of?-aminobutyric acid aminotransferase,together with glutamate decarboxylase and succinate semialdehyde dehydrogenase forming?-aminobutyric acid metabolic bypass,which completes the TCA cycle in cyanobacteria.In this study,the N-acetylornithine aminotransferase gene(slr1022 gene)was successfully cloned from Synechocystis sp.PCC6803 genomic DNA and linked to the p ET-28a expression vector,and then the constructed plasmid was transformed into E.coli BL21(DE3).The recombinant Slr1022 protein was purified by Ni-NTA affinity chromatography after the expressing strain was induced by IPTG.Its kinetic characterization was carried out by ultraviolet spectrophotometry and enzyme coupling method in detail.The results showed that the optimal p H of the enzyme was 8.5,optimal reaction temperature was 30?,Mg²⁺had the best activation effect,the values of K_m and k_cat with Ac Orn were 0.12±0.015 mmol/L and 2.31 s^-1,and the value of K_m and k_cat with?-KG were 0.037±0.004 mmol/L and 2.50 s^-1,respectively.Through multisequence alignment and homology modeling methods,the amino acid residues located near the substrate or cofactor were found and mutated in this study.Then the mutant protein was purified and kinetically characterized.The results showed that Lys280 and Asp251 residues were the key amino acids in N-acetylornithine aminotransferase,in which the?-amino group of Lys280 residue bound with PLP covalently through Schiff base to form internal aldimine initiating the catalytic reaction,and in which Asp251 residue formed a hydrogen bond or salt bridge with the protonated nitrogen in the PLP pyridine ring to stabilize the protonated nitrogen.Glu223 residue was involved in the substrate binding,Thr308 and Q254 residues were involved in the catalytic reaction of N-acetylornithine aminotransferase and Tyr39,Arg163 and Arg402 residues played important roles in the substrate recognition and catalytic process of the reaction.Finally,the catalytic mechanism of N-acetylornithine aminotransferase was deduced based on the results of mutation experiments and the studies on other transaminases of the same family.