Catalytic Functions And Mechanisms Of Key Enzymes In The Pathway Of ?-Ketoglutaric Acid To Succinic Acid In Cyanobacteria

The tricarboxylic acid cycle is a common metabolic pathway in aerobic organisms.However,the tricarboxylic acid cycle in cyanobacteria is different from the canonical tricarboxylic acid cycle due to the lack of?-ketoglutarate dehydrogenase.In this thesis,the proteins encoded by the sll1981 gene of Synechocystis sp.PCC6803 and the all3556gene of Anabaena PCC7120 were biochemically characeterized in detail.The key enzymes involved in the metabolic pathway of?-ketoglutarate to succinic acid in cyanobacteria are?-ketoglutarate decarboxylase and succinic semialdehyde dehydrogenase.?-Ketoglutarate decarboxylase catalyzes the non-oxidative decarboxylation of?-ketoglutarate to succinic semialdehyde.Bioinformatics analysis indicated that the sll1981 gene in Synechocystis sp.PCC6803 may encode?-ketoglutarate decarboxylase.In this study,the sll1981 gene was cloned from the genome of Synechocystis sp.PCC6803 and ligated into the pCold-TF vector and transformed into E.coli BL21(DE3)cells.The recombinant sll1981 protein was purified by affinity chromatography.Its substrate specificity and kinetic parameters against pyruvate,D-glucose-6-phosphate and?-ketoglutarate were characterized.The results showed that the sll1981 protein was extremely unstable,the trigger factor(TF)and the tig protein expressed by the chaperone plasmid pTf16 could increase the solubility of sll1981 protein.Steady-state kinetics indicated that the sll1981 protein was a bifunctional enzyme,whose activity as?-ketoglutarate decarboxylase was 444 times of that as acetolactate synthase.Meanwhile,sll1981 protein didn't have inositol-1-phosphate synthesis activity,although it was previously reported to be an inositol-1-phosphate synthase.Succinic semialdehyde dehydrogenase catalyzes the formation of succinic acid from succinic semialdehyde.The homologous sequence alignment revealed that the all3556gene from Anabaena sp.PCC7120 was likely to encode a succinic semialdehyde dehydrogenase.In this study,the pET28a-all3556 expression plasmid was constructed and transformed into E.coli BL21(DE3)cells for expression,and then homogenous all3556 protein was purified by affinity chromatography.Enzyme kinetics showed that all3556 protein was an NADP~+-dependent succinic semialdehyde dehydrogenase which could be inhibited by its substrate.Its substrate inhibition concentration was 0.1 mmol/L.In addition,the research found that the Ser157 residue determined the selectivity of the cofactor.When the Ser157 residue was mutated to Glu or Pro,the preferred cofactor shifted from the original NADP~+to NAD~+.At the same time,it was found that ApSSADH activity was greatly reduced or even inactive after mutating the residues of the catalytical active center.Bioinformatics analysis and model structure indicated that these amino acid residues were highly conserved.The catalytic mechanism of ApSSADH was proposed based on the kinetic parameters of wild type and mutant proteins.