Crystalline green 5‐hydroxyvaleryl‐CoA dehydratase from Clostridium aminovalericum

A green enzyme from Clostridium aminovalericum with valeryl‐CoA dehydrogenase activity was purified to homogeneity (169 ± 3 kDa) and crystallized. By SDS/PAGE, one type of subunit (42 kDa) was detected indicating a homotetrameric structure. The unusual ultraviolet/visible spectrum of the green enzyme (maxima at 394 nm, 438 nm and 715 nm) was converted to a normal flavoprotein spectrum either by reduction with dithionite and reoxidation under air, or by removal of the prosthetic group at pH 2 and reconstitution with FAD (not FMN). Besides FAD (4 mol/169 kDa), the enzyme contained 4 mol of a CoA ester which was similar but not identical to 5‐hydroxy‐2‐pentenoyl‐CoA. The reconstituted holoenzyme as well as the native green enzyme, but not the apoenzyme, catalysed the reversible dehydration of 5‐hydroxyvaleryl‐CoA to 4‐pentenoyl‐CoA in the absence of an external electron acceptor. In its presence (preferentially ferricenium ion), the green or yellow enzyme catalysed the formation of ( E )‐5‐hydroxy‐2‐pentenoyl‐CoA and 2,4‐pentadienoyl‐CoA either from 4‐pentenoyl‐CoA or from 5‐hydroxyvaleryl‐CoA. The reversible hydration of 2,4‐pentadienoyl‐CoA to ( E )‐5‐hydroxy‐2‐pentenoyl‐CoA was mediated by both enzymes as well as by the apoenzyme in the absence of FAD. Hydration of 4‐pentenoate in 2 H 2 O yielded optically active 5‐hydroxy[2,4‐ 2 H 2 ]valerate by the combined action of 5‐hydroxyvalerate CoA‐transferase, the green dehydratase and catalytical amounts of acetyl‐CoA. The data show that the reversible hydration of the isolated double bond of 4‐pentenoyl‐CoA to 5‐hydroxyvaleryl‐CoA, which apparently violates the Markovnikov rule, is preceded by oxidation to 2,4‐pentadienoyl‐CoA. The latter compound, a vinyl analogue of 2‐enoyl‐CoA, is then easily hydrated to ( E )‐5‐hydroxy‐2‐pentenoyl‐CoA and finally reduced to 5‐hydroxyvaleryl‐CoA.