Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation 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-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.
Scherf
U
U
Laboratorium für Mikrobiologie des Fachbereiches Biologie der Philipps-Universität, Marburg, Germany.
Söhling
B
B
Gottschalk
G
G
Linder
D
D
Buckel
W
W
D000595:Amino Acid Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D003013:Clostridium / Q000201:enzymology*; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D017421:Sequence Analysis; D013386:Succinates / Q000378:metabolism*; D019802:Succinic Acid; D013455:Sulfur / Q000378:metabolism
D013386:Succinates; D005182:Flavin-Adenine Dinucleotide; D000431:Ethanol; D013455:Sulfur; D019802:Succinic Acid; D007501:Iron; C066370:4-hydroxybutyryl-CoA dehydratase; D006836:Hydro-Lyases; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C023288:vinylacetyl-CoA delta-isomerase