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  • Title: Crystalline green 5-hydroxyvaleryl-CoA dehydratase from Clostridium aminovalericum.
    Author: Eikmanns U, Buckel W.
    Journal: Eur J Biochem; 1991 May 08; 197(3):661-8. PubMed ID: 2029896.
    Abstract:
    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 2H2O yielded optically active 5-hydroxy[2,4-2H2]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.
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