• Title/Summary/Keyword: ${\alpha}$-Keto acid dehydrogenase complexes

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Asparagine-473 Residue Is Important to the Efficient Function of Human Dihydrolipoamide Dehydrogenase

  • Kim, Hak-Jung
    • BMB Reports
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    • v.38 no.2
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    • pp.248-252
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    • 2005

  • Dihydrolipoamide dehydrogenase (E3) catalyzes the reoxidation of dihydrolipoyl moiety of the acyltransferase components of three $\alpha$-keto acid dehydrogenase complexes and of the hydrogen-carrier protein of the glycine cleavage system. His-457 of Pseudomonas putida E3 is suggested to interact with the hydroxyl group of Tyr-18 of the other subunit and with Glu-446, a component in the last helical structure. To examine the importance of the suggested interactions in human E3 function, the corresponding residue of human E3, Asn-473, was substituted to Leu using site-directed mutagenesis. The E3 mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 37-fold, indicating that Asn-473 residue was important to the efficient catalytic function of human E3. Its slightly altered spectroscopic properties implied that small conformational changes could occur in the E3 mutant.

  • https://doi.org/10.5483/BMBRep.2005.38.2.248 인용 PDF

Activity of Human Dihydrolipoamide Dehydrogenase Is Largely Reduced by Mutation at Isoleucine-51 to Alanine

  • Kim, Hak-Jung
    • BMB Reports
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    • v.39 no.2
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    • pp.223-227
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    • 2006

  • Dihydrolipoamide dehydrogenase (E3) belongs to the pyridine nucleotide-disulfide oxidoreductase family including glutathione reductase and thioredoxin reductase. It catalyzes the reoxidation of dihydrolipoyl moiety of the acyltransferase components of three $\alpha$-keto acid dehydrogenase complexes and of the hydrogen-carrier protein of the glycine cleavage system. Isoleucine-51 of human E3, located near the active disulfide center Cys residues, is highly conserved in most E3s from several sources. To examine the importance of this highly conserved Ile-51 in human E3 function, it was substituted with Ala using site-directed mutagenesis. The mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 100-fold, indicating that the conservation of the Ile-51 residue in human E3 was very important to the efficient catalytic function of the enzyme. Its altered spectroscopic properties implied that conformational changes could occur in the mutant.

  • https://doi.org/10.5483/BMBRep.2006.39.2.223 인용 PDF