SUCCINATE-ETHANOL FERMENTATION IN CLOSTRIDIUM-KLUYVERI - PURIFICATIONAND CHARACTERIZATION OF 4-HYDROXYBUTYRYL-COA DEHYDRATASE VINYLACETYL-COA DELTA(3)-DELTA(2)-ISOMERASE/

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybu tyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under stric tly anaerobic conditions to over 95% homogeneity and had a specific ac tivity of 123 nkat mg(-1). The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethano l by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate v ia 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m = 59 kDa/subunit), containing 2 +/- 0.2 mol FAD, 13 .6 +/- 0.8 mol Fe and 10.8 +/- 1.2 mol inorganic sulfur. The enzyme wa s irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate (III) . The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. amin obutyricum. Moreover, the N-terminal sequences of the dehydratases fro m both organisms were found to be 63% identical.