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

Abstract

2,4‐Pentadienoyl‐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/visible‐spectrum 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‐pentadienoyl‐CoA 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 ‐isomerase activity (1.2 nkat/mg), the only product of the reduction was 3‐pentenoyl‐CoA rather than 2‐pentenoyl‐CoA. Copyright © 1991, Wiley Blackwell. All rights reserved