Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2âoxoglutarate dehydrogenase complex
2000
Koike, Kichiko | Suematsu, Takashi | Ehara, Masahiko
Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2âoxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 aminoâacid residues) and a mature protein (387 residues, Mrâ=â41â534). Recombinant porcine E2o (rPE2o) (residues 1–387), Câ and Nâterminal truncated PE2os, and siteâdirected mutant PE2os were overexpressed in Escherichia coli via the expression vector pETâ11d and purified. The succinyltransferase activity of the rPE2o was about 2.2âfold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cubeâlike structure very similar to that of the native PE2o. Deletion of five aminoâacid residues from the Câterminus resulted in a complete loss of both enzymatic activity and formation of the cubeâlike structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the Câterminal leucine triplet (Leu383–384–385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383–384–385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5âfold reduction in kcat, with little change in Km values for dihydrolipoamide and succinylâCoA. However, selfâassembly was not affected. These data indicate that Ser306, Asp362 and the Leu383–384–385 triplet are important residues in both the selfâassembly and catalytic mechanism of PE2o.
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