Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824).
Two butanol dehydrogenases with different cofactor requirements and different pH ranges have been detected in Clostridium acetobutylicum ATCC 824. The NADH-dependent butanol dehydrogenase (NADH-BDH) was purified to near homogeneity and characterized. One striking feature of the enzyme is that Zn2+ was needed to obtain a significant recovery during purification. The enzyme was a dimer composed of two subunits with subunit molecular mass of 42 kDa and a native molecular mass of 82 +/- 2 kDa. The kinetics were studied in the direction of the reduction of butyraldehyde. Inhibition studies with S-NADH and butanol indicate that the NADH-BDH follows an ordered bibi mechanism with kinetic constants of 4.86 s-1, 0.18 mM, and 16 mM for Kcat, KNADH, and Kbutyraldehyde, respectively. Activity in the reverse direction was 50-fold lower than that in the forward direction. The NADH-BDH had higher activity with longer chained aldehydes and was inhibited by metabolites containing an adenine moiety.
Welch
R W
RW
Department of Biochemistry, Rice University, Houston, Texas 77005.
Rudolph
F B
FB
Papoutsakis
E T
ET
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
D000429:Alcohol Oxidoreductases; C060833:butanol dehydrogenase