Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea.
Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 degrees C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO(2), H(2), L-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 +/- 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 +/- 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe(2+). The enzyme was thermostable with a half-life of about 10 h at 70 degrees C, and its catalytic activity increased along with the rise of temperature up to 95 degrees C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K (m) values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.
Ying
Xiangxian
X
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1 Canada.
Wang
Ying
Y
Badiei
Hamid R
HR
Karanassios
Vassili
V
Ma
Kesen
K
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D010100:Oxygen / Q000494:pharmacology; D013379:Substrate Specificity
D007501:Iron; D000426:Alcohol Dehydrogenase; D010100:Oxygen