• 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.

• BMB Reports
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• v.35 no.4
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• pp.437-441
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• 2002

Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family. Thr residues are highly conserved. They are at the active site disulfide-bond regions of most E3s and other oxidoreductases,. The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD. However, several prokaryotic E3s have Val instead of Thr. To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis. The mutant’s E3 activity showed about a 2.2-fold decrease. Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.

• 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.