Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, M-r = 41 534). Recombinant porcine E2o (rPE2o) (residues 1-387), C- and N- terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The su ccinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained s howed a cube-like structure very similar to that of the native PE2o. Deleti on of five amino-acid residues from the C-terminus resulted in a complete l oss of both enzymatic activity and formation of the cube-like structure, bu t the deletion of only the last two residues had no effect on either functi on, suggesting the important roles of the C-terminal leucine triplet (Leu38 3-384-385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Al a in the putative active site, and Leu383-384-385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold re duction in k(cat), with little change in K-m values for dihydrolipoamide an d succinyl-CoA. However, self-assembly was not affected. These data indicat e that Ser306, Asp362 and the Leu383-384-385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.