A green 2,4‐pentadienoyl‐CoA reductase from Clostridium aminovalericum

Abstract

2,CPentadienoyl-CoA reductase from Clostridium aminovalericum was purified to homogeneity (170 -182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visiblespectrum showed absorption maxima at 270,402 and 715 nm as well as shoulders at 278, 360,450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/ subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two.The green enzyme catalysed the oxidation of (E)-3-pentenoyl-CoA or (E)-3-hexenoyl-CoA to 2,4-pentadienoylCoA or 2,4-hexenoyl-CoA, respectively. 2-Pentenoyl-CoA or 4-pentenoyl-CoA were not oxidised. Meldola blue (8-dimethylamino-2,3-benzophenoxazine) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride ( V = 26 nkat/mg protein) or ferricenium hexafluorophosphate ( V = 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4-pentadienoyl-CoA ( V = 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl-CoA A-isomerase activity (1.2 nkat/mg), the only product of the reduction was 3-pentenoyl-CoA rather than 2-pentenoylCoA.

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

“…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]. 3-Pentenoyl-CoA is isomerized to 2-pentenoyl-CoA, which disproportionates to acetate, propionate and valerate [30] …”

Crystallization and preliminary X-ray diffraction study of the flavoenzyme 2,4-pentadienoyl-CoA reductase from Clostridium aminovalericum

“…The precipitate was discarded and the remaining solution applied to a phenylsepharose HP column. For continuous routine measurements 3-pentenoyl-CoA served as substrate and Meldola blue (8-dimethylamino-2,3-benzophenoxazine) together with INT [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride] was used as electron acceptor (kcat = 16s -1) (Eikmanns & Buckel, 1991c). The most active colored fractions were pooled and purified further by size-exclusion chromatography on Superdex 200 followed by anion-exchange chromatography.…”

“…Incubation of the enzyme with an excess of FAD results in 0.9mol FAD/subunit and the specific activity increases twofold. The unusual UV-visible spectrum of this greenish enzyme (absorption maxima at 404 and 707 nm, shoulder at 371 and 460nm) can be converted to that of a normal yellow flavoprotein (absorbance maxima at 370 and 450nm) by removal of the prosthetic group followed by reconstitution with FAD, but not by reduction with dithionite followed by reoxidation by air (Eikmanns & Buckel, 1991c). Neither of these treatments abolished enzymic activity, indicating that the green charge-transfer complex was not an intermediate in the reaction pathway.…”

“…The resulting 5-hydroxyvalerate is activated to 5-hydroxyvaleryl-CoA by the enzyme CoA transferase, which has been purified (Eikmanns & Buckel, 1991a). The next three reaction steps are catalyzed by two green enzymes, the bifunctional 5-hydroxyvalerylCoA dehydrogenase/dehydratase, which has recently been crystallized (Eikmanns & Buckel, 1991b;Eikmanns, Buta, Buckel & Pai, 1994), and 2,4-pentadienyl-CoA reductase (Eikmanns & Buckel, 1991c). The product, 3-pentenoyl-CoA, isomerizes to 2-pentenoyl-CoA which then disproportionates to valerate as well as to acetate and propionate.…”

Aromatizing Cyclohexa-1,5-diene-1-carbonyl-Coenzyme A Oxidase

“…The UV-visible spectrum of DCO indeed provided initial evidence that the enzyme is a flavo/FeS protein, and it was shown that 1,5-dienoyl-CoA reduced these cofactors. DCO, old yellow enzymes, and 2,4-dienoyl-CoA reductases use ␣,␤-unsaturated carbonyl compounds as electron donors (30,(32)(33)(34)(35)(36)(37). Whereas the latter two reduce their substrates most often with NADPH, only DCO catalyzes an irreversible oxidation of its substrate.…”