Purification and properties of an iron‐sulfur and FAD‐containing 4‐hydroxybutyryl‐CoA dehydratase/vinylacetyl‐CoA3‐2‐isomerase from Clostridium aminobutyricum

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Abstract
4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of γ-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 μM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA 3-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 irrevesible inactivation. Concomitantly the vinylacetyl-CoA -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 hexacyanofer-rate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration 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 enzymes [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-xybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.