| Cyanobacteria have recently been found to contain a bypassing metabolic pathway ofα-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase,which can convertα-ketoglutarate to succinic acid and complete the tricarboxylic acid cycle(TCA cycle)in cyanobacteria.However,there is no detailed biochemical characterization of these two enzymes in this pathway.BLAST analysis revealeds that the gene cce4227 in Cyanothece sp.ATCC51142 was likely to encode an α-ketoglutarate decarboxylase,although the cce4227 gene was proposed to encode the large-subunit of acetolactate synthase in NCBI.Therefore,the recombinant plasmid pET28a-cce4227 was constructed by molecular cloning,and the co-expression system was formed with molecular chaperone pTf16.The recombinant cce4227 protein was purified by Ni-NTA affinity chromatography,and its catalytic function was identified.At the same time,its kinetic characterization was carried out by ultraviolet spectrophotometry and enzyme coupling method.The specificity ofα-ketoglutarate decarboxylase to various substrates was determined.The results demonstrated that cce4227 protein was an α-ketoglutarate decarboxylase,although it also possessed weak activity of acetolactate synthase,which was only 1/400 that ofα-ketoglutarate decarboxylase.Biochemical characterization of α-ketoglutarate decarboxylase showed that the optimum pH of the enzyme was 7.5;the optimum reaction temperature and the best metal ions to activate were 40℃ and Mg2+respectively.In addition,Km value of α-ketoglutarate acid was 0.67 ± 0.14 mmol/L,kcat value was 2.53 ±0.16 s-1;Km value of TPP is 0.15±0.01 mmol/L,the kcat value is 2.56 ± 0.06 s-1,α-ketoglutarate acid was the specific substrate.Through three-dimensional structure simulation,amino acid residues located near TPP were found and mutated in this study.The mutant proteins were purified and kinetic characterized.The results showed that L467,Y465 and D435 residues were involved in the binding with the substrate α-ketoglutarate acid,and especially D435 residue was also involved in the binding with the cofactor TPP.In addition,the residues of Y465,D435 and K386 played an important role of acid base catalysis in the catalytic process ofα-ketoglutarate decarboxylase.Finally,a possible catalytic mechanism of α-ketoglutarate decarboxylase was suggested based on the results of kinetics and previous results of various ketoacids decarboxylases,... |
