title: Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.
abstract: The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.
authors.affiliation: Biochemistry Department, Michigan State University, East Lansing 48824.
authors.forename: D
authors.initials: D
authors.lastname: Burdette
authors.forename: J G
authors.initials: JG
authors.lastname: Zeikus
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology*; D007700:Kinetics; D010084:Oxidation-Reduction; D013869:Thiolester Hydrolases / Q000378:metabolism*
chemical_list: D000105:Acetyl Coenzyme A; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+); D013869:Thiolester Hydrolases