title: Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri.
abstract: The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.
authors.affiliation: Graduate School of Bioresources, Mie University, Tsu 514-8507, Japan.
authors.forename: Saikat
authors.initials: S
authors.lastname: Chakraborty
authors.forename: Makiko
authors.initials: M
authors.lastname: Sakka
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kimura
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sakka
mesh_terms: D047010:Clostridium kluyveri / Q000201:enzymology* / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D007700:Kinetics; D009245:NADH Dehydrogenase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D011994:Recombinant Proteins; D009245:NADH Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase