Abstract:[Background] The cyanobacterial tricarboxylic acid cycle, which produces succinic acid, is different from that of other species. The existences of α-ketoglutarate decarboxylase and succinic semialdehyde dehydrogenase in cyanobacterium make its tricarboxylic acid cycle complete. Succinic semialdehyde dehydrogenases that catalyze the conversion of succinic semialdehyde to succinic acid, exist in cyanobacteria extensively. [Objective] Clone, express and purify the protein encoded by the cce4228 gene of Cyanothece sp. ATCC51142, and characterize its biochemical properties in vitro. [Methods] cce4228 gene was cloned into pET-28a vector, overexpression of recombinant cce4228 protein was induced by isopropyl β-D-thiogalactoside (IPTG) in E. coli BL21(DE3) and then the protein was purified by Ni-NTA affinity chromatography. The biochemical properties of cce4228 protein were characterized by spectrophotometric and bioinformatics methods. [Results] An expression plasmid pET-28a-cce4228 was constructed and cce4228 protein was overexpressed in soluble form in E. coli BL21(DE3). Recombinant cce4228 protein with purity higher than 90% was obtained. Steady-state kinetic and bioinformatic studies demonstrated that cce4228 protein was a NADP+-dependent succinic semialdehyde dehydrogenase. [Conclusion] The cce4228 gene in Cyanothece sp. ATCC51142 encodes a succinic semialdehyde dehydrogenase, which prefers to use NADP+ rather than NAD+ as its cofactor. These results will provide basis to understand the structure-function relationship and catalytic mechanism of cce4228 protein further.
