Biochemical and Structural Insights into a Thiamine Diphosphate-Dependent α-Ketoglutarate Decarboxylase from Cyanobacterium NIES-843.

α-Ketoglutarate decarboxylase is a crucial enzyme in the tricarboxylic acid cycle of cyanobacteria, catalyzing the non-oxidative decarboxylation of α-ketoglutarate to produce succinate semialdehyde and CO. The decarboxylation process is reliant on the cofactor of thiamine diphosphate. However, this enzyme's biochemical and structural properties have not been well characterized. In this work, two α-ketoglutarate decarboxylases encoded by and genes from NIES-843 (MaKGD) and NIES-4325 (MiKGD), respectively, were overexpressed and purified by using an expression system. It was found that MaKGD exhibited 9.2-fold higher catalytic efficiency than MiKGD, which may be attributed to the absence of glutamate decarboxylase in NIES-843. Further biochemical investigation of MaKGD demonstrated that it displayed optimum activity at pH 6.5-7.0 and was most activated by Mg. Additionally, MaKGD showed substrate specificity towards α-ketoglutarate. Structural modeling and autodocking results revealed that the active site of MaKGD contained a distinct binding pocket where α-ketoglutarate and thiamine diphosphate interacted with specific amino acid residues via hydrophobic interactions, hydrogen bonds and salt bridges. Furthermore, the mutagenesis study provided strong evidence supporting the importance of certain residues in the catalysis of MaKGD. These findings provide new insights into the structure-function relationships of α-ketoglutarate decarboxylases from cyanobacteria.