Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum.
4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.
Scherf
U
U
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität Marburg, Germany.
Buckel
W
W
D000214:Acyl Coenzyme A / Q000378:metabolism; D000693:Anaerobiosis; D019748:Carbon-Carbon Double Bond Isomerases; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006836:Hydro-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000032:analysis
D000214:Acyl Coenzyme A; D005182:Flavin-Adenine Dinucleotide; D013455:Sulfur; D007501:Iron; C066370:4-hydroxybutyryl-CoA dehydratase; D006836:Hydro-Lyases; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C023288:vinylacetyl-CoA delta-isomerase