Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA ?3-?2-isomerase

Abstract

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-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 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 was 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. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.

Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO 2 fixation

“…This possibility raised our interest, because closely related clostridial 4-hydroxybutyryl-CoA dehydratases had been reported to be extremely oxygen-sensitive (28,29). The presence of the oxygen-tolerant 4-hydroxybutyryl-CoA dehydratase in Thaumarchaeota is a biochemical requirement for the operation of the HP/HB cycle under aerobic conditions.…”

Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota

“…The incubation of cell extracts with air for 2 h did not result in any significant decrease of 4-hydroxybutyryl-CoA dehydratase and fumarate reductase activity. Interestingly, purified 4-hydroxybutyryl-CoA dehydratase from Clostridium kluyveri was more oxygen-labile and had a half-life of approximately 30 min (Scherf et al, 1994). We have presented evidence that the strictly anaerobic S. azoricus fixes CO 2 via the 3-hydroxypropionate/4-hydroxybutyrate cycle.…”

“…The enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle tolerate oxygen. Although 4-hydroxybutyryl-CoA dehydratase is inactivated by oxygen (Scherf et al, 1994), it may be sufficiently stable at low oxygen tensions to maintain activity, especially in the protected environment of the cell. In contrast, the oxygen sensitivity of some of the enzymes and electron carriers of the dicarboxylate/4-hydroxybutyrate cycle such as pyruvate synthase and ferredoxin seems to restrict this cycle to organisms growing under anoxic or microoxic conditions.…”

The genome ofClostridium kluyveri, a strict anaerobe with unique metabolic features

“…Ethanol-succinate fermentation to acetate and butyrate involves succinyl-CoA, succinate semialdehyde, 4-hydroxybutyrate, and 4-hydroxybutyryl-CoA as intermediates, which are not involved in ethanol-acetate or crotonate fermentations. Two specific CoA transferases (Cat1 and Cat2), NAD-dependent succinate semialdehyde dehydrogenase (SucD), NAD-dependent 4-hydroxybutyrate dehydrogenase (4Hbd), and 4-hydroxybutyryl-CoA dehydratase/isomerase (AbfD) have been characterized (42). The genes for these enzymes are clustered (SI Table 2), and their expression is induced by succinate (43,44).…”