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, Mr = 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 succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal 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 8.5-fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383-384-385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.
Koike
K
K
Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki University School of Medicine, Japan. Kichiko.Koike@mb5.seikyou.ne.jp
Suematsu
T
T
Ehara
M
M
D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D018076:DNA, Complementary; D009841:Oligonucleotides; D011994:Recombinant Proteins; D007655:Ketoglutarate Dehydrogenase Complex; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase