Purification and Characterization of Crotonase from Clostridium acetobutylicum

Abstract An enzyme catalyzing the reversible hydration of crotonylCoA has been obtained in homogeneous form from Clostridium acetobutylicum. The bacterial hydrase has a molecular weight of 158,000 ± 3,000 as determined by sedimentation equilibrium. In contrast, the enzyme has a molecular weight of approximately 43,000 in 6 m guanidine hydrochloride in either the presence or absence of reducing agent. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate yields a molecular weight of 40,000, also about one-fourth that of the native enzyme. Unlike bovine liver crotonase, the bacterial enzyme is specific for short chain fatty acyl-CoA substrates and is sensitive to high concentrations of crotonyl-CoA. It requires a complete coenzyme A thioester substrate for efficient catalysis. It is concluded that crotonase from C. acetobutylicum is composed of 4 subunit polypeptide chains, with each chain containing about 370 residues devoid of any attached carbohydrate, and that the 4 subunits of the native enzyme are combined through noncovalent interactions. There are, however, a number of similarities between the bacterial and mammalian enzymes, which suggest that the two may be structurally related.