Crystalline green 5‐hydroxyvaleryl‐CoA dehydratase from Clostridium aminovalericum

Abstract

A green enzyme from Clostridium aminovalericum with valeryl-CoA dehydrogenase activity was purified to homogeneity (169 5 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 mo1/169 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 (@-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 (@-5-hydroxy-2-pentenoylCoA was mediated by both enzymes as well as by the apoenzyme in the absence of FAD. Hydration of 4-pentenoate in 2 H z 0 yielded optically active 5-hydro~y[2,4-~H~]valerate by the combined action of 5-hydroxyvalerate CoAtransferase, 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)-S-hydroxy-2-pentenoyl-CoA and finally reduced to 5-hydroxyvaleryl-Co A.

Unusual DAhydrations in anaerobic bacteria: considering ketyls (radical anions) as reactive intermediates in enzymatic reactions

“…4). This transient introduction of a double bond activates the 7-hydrogen to be easily removed as a proton [32]. In vivo 2,4-pentadienoyl-CoA is released from the enzyme and reduced in a different manner by 2,4-addition of a proton and a hydride to 3-pentenoyl-CoA, which is catalysed by a different also FAD-containing greenish reductase [33].…”

“…Proposed mechanism for 1,2-propandiol dehydratase involving a ketyl as intermediate.shows significant similarities to short and medium chain acyl-CoA dehydrogenases (U. Eikmanns, C. Buta and W.Buckel, unpublished). This enzyme and 2,4-pentadienoyl-CoA reductase have been crystallized, and their 3-dimensional structures are currently under investigation[32,34,35]•…”

Acryloyl‐CoA reductase from Clostridium propionicum

“…The yellow supernatant was analysed by HPLC on LiChroCart RP‐18 (250 mm × 4 mm; LiChrosphere; 5 µm; Merck) by using isocratic elution with 50 m m aqueous ammonium formate/methanol (75 : 25, v/v) at a flow rate of 1 mL·min −1 . Fractions containing FAD or FMN were detected by their A 269 and coelution with standards [32]. For quantitative determination of the flavin content, a concentrated stock solution was used containing 114 µ m acryloyl‐CoA reductase (17 mg protein·mL −1 ), 50 m m potassium phosphate, pH 7.5, and 10 µ m FAD.…”

Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems

“…This pathway recognizes both cholic and chenodeoxycholic acid and may be the major, if not sole, bile acid 7α‐dehydroxylation pathway in human intestinal bacteria [60, 61]. Similar biochemical reactions appear to be carried out by Clostridium aminovalericum during fermentation of 5‐aminovalerate [62]. The key enzyme, 5‐hydroxyvaleryl‐CoA dehydratase, catalyzes the oxidation of 5‐hydroxyvaleryl‐CoA to 5‐hydroxy‐2‐pentenoyl‐CoA, the dehydration to 2,4‐pentadienoyl‐CoA and the reduction to 4‐pentenoyl‐CoA.…”