Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.
The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases.
Palosaari
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Department of Microbiology, University of Minnesota, Minneapolis 55455.
Rogers
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P
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D012293:Rifampin / Q000494:pharmacology
D000214:Acyl Coenzyme A; D000440:Butanols; C024343:butyryl-coenzyme A; D000431:Ethanol; D000105:Acetyl Coenzyme A; D000445:Aldehyde Oxidoreductases; C056375:butyraldehyde dehydrogenase (Coenzyme A-linked); D012293:Rifampin