Study On Biochemical Characteristics And Catalytic Mechanism Of α-Ketoglutarate Decarboxylase From Microcystis Aeruginosa

α-Ketoglutarate decarboxylase catalyzes the non-oxidative decarboxylation ofα-ketoglutarate to succinate semialdehyde and CO₂ depending on the activation of the cofactor TPP and divalent metal ions.This enzyme is a key enzyme in the KGD bypass metabolic pathway in cyanobacteria.In this study,the MAE＿06010 and Mi Abw＿01735genes encodingα-ketoglutarate decarboxylases derived from Microcystis aeruginosa and the molecular chaperone p Tf16 were used to form a co-expression system,which were transferred into Escherichia coli BL21（DE3）strain for inducible expression.Abundant soluble proteins were purified by nickel affinity chromatography,and their kinetics and catalytic function were characterized by microplate reader and enzyme coupling method.The substrate specificity ofα-ketoglutarate decarboxylase was also detected.The results showed that MAE＿06010 protein and Mi Abw＿01735 protein had bothα-ketoglutarate decarboxylase and acetolactate synthase activity,whereas the activity of acetolactate synthase was only 1/200 of that ofα-ketoglutarate decarboxylase.The results of biochemical characterization ofα-ketoglutarate decarboxylase showed that the optimum p H of the enzyme was 7.0,the optimum reaction temperature was 40℃,the activation effect of Mg²⁺and Mn²⁺was the best,and it had substrate specificity forα-ketoglutarate.The K_m value of the MAE＿06010 for the substrateα-ketoglutarate was 0.87±0.23 mmol/L,and the k_cat was 7.8 s^-1;the K_m value for the cofactor TPP was 0.0056±0.0007 mmol/L,and the k_cat was 2.9 s^-1.The K_m value of the Mi Abw＿01735 for the substrateα-ketoglutarate was 4.47±2.63 mmol/L,and the k_cat was 4.3 s^-1;the K_m value for the cofactor TPP was0.026±0.006 mmol/L,and the k_cat was 3.2 s^-1.In this study,by utilizing multiple sequence alignment and homology modeling of the MAE＿06010 protein,the amino acid residues near the cofactor TPP were found and then site-directed mutagenesis was carried out,and the mutant protein was purified for kinetic characterization.The results showed that almost all the mutated amino acid residues affected the binding of the enzyme to TPP.The D435 residue was involved in the binding of the substrateα-ketoglutarate and was used as an acid-base catalyst to affect the enzymatic reaction process;residues Y465,L467,M410 and D462 were involved in the binding of the substrateα-ketoglutarate;residue M410 also supported the“V”configuration of TPP;residue E50 catalyzed the formation of ylides from TPP;and residue D462 acted as an electronic intermediary to participate in the catalytic process.Finally,the possible catalytic mechanism ofα-ketoglutarate decarboxylase was deduced from the results of mutation studies and studies of other ketoacid decarboxylases in the same family.Under the action of E50 residues,TPP protons form Yelid structure,and then attack the substrate to generate CO2 and enamine intermediates.The D462 residue acts as a proton donor and acceptor and is deprotonated to form the product succinate hemaldehydes.