| Succinic semialdehyde dehydrogenase is widely present in bacteria,fungi,plants and mammals,and it plays a vital role in various detoxification and physiological metabolic pathways in various organisms.Succinic semialdehyde dehydrogenase relies on the cofactor NAD(P)+to catalyze the conversion of succinic semialdehyde(SSA)to succinic acid(SA).The participation of succinic semialdehyde dehydrogenase in the α-ketoglutarate(α-KG)and y-aminobutyric acid(GABA)bypasses completes the tricarboxylic acid(TCA)cycle in cyanobacteria,that is,the presence of succinic semialdehyde dehydrogenase enriches TCA cycle.In this study,the succinic semialdehyde dehydrogenase gene gene)was successfully cloned from Cyanothece sp.ATCC51142 genomic DNA and successfully constructed it into the pET-28a prokaryotic expression vector.It was found that the expression level and solubility of cce4228 protein in E.coli were excellent.Subsequently,homogeneous and stable cce4228 protein was obtained by Ni-NTA affinity chromatography and the kinetics were characterized in detail.The results showed that the cce4228 protein was a succinate semialdehyde dehydrogenase which preferred the cofactor NADP+,Biochemical characterization demonstrated that cce4228 protein existed as a dimer in its natural state,the optimal pH of the cce4228 protein was 9.0,and Mg2+ was the best activator among tested metal ions for cce4228 protein.Meanwhile,the cce4228 protein displayed obvious substrate inhibition phenomenon,since SSA occupied the binding pocket of NADP+with cce4228.In addition,model structure of cce4228 protein with SSA and NADP+was simulated and site-directed mutants was constructed and biochemical characterization of each mutant protein was performed.The results showed that Ser157 residue in cce4228 protein determined its cofactor preference,and Lys154 residue assists Ser157 residue to perform its function.At the same time,Mg2+located between Ser157 and Lys154 residues,the Arg139 residue played a key role in maintaining the spatial conformation of the substrate SSA and cofactor NADP+,Cys262 and Glu228 residues were the key amino acid residues for cce4228 protein to play a catalytic vital role,Asn131 residue stabilized the intermediates in the catalytic process through hydrogen bonding. |
