Lipoamide dehydrogenase from streptomyces seoulensis: biochemical and genetic properties.
Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9. 5%. The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit. The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm. Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH. Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase. The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier. The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism. The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1, 4-naphthosemiquinone radical anion. The quinone-reducing activity of the enzyme was dramatically inhibited by NAD+, indicating the involvement of four-electron reduced form. The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences. The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes. The gene was named lpd. All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.
Youn
H
H
Department of Microbiology, College of Natural Sciences, and Research Centre for Molecular Microbiology, Seoul National University, Seoul 151-742, South Korea.
Kwak
J
J
Youn
H D
HD
Hah
Y C
YC
Kang
S O
SO
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000494:pharmacology; D009285:Naphthoquinones / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009285:Naphthoquinones; D009243:NAD; D008058:Dihydrolipoamide Dehydrogenase; C035342:1,4-naphthoquinone