Purification and Some Properties of a Hitherto‐Unknown Enzyme Reducing the Carbon‐Carbon Double Bond of α,β‐Unsaturated Carboxylate Anions

Abstract

2‐Enoate‐reductase, a previously unknown soluble enzyme is present in Clostridium kluyveri and another Clostridium species growing on (E)‐2‐butenoate. From the latter the reductase was purified 88‐fold with an overall yield up to 74%. The enzyme was pure as judged by polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate as well as by isoelectric focusing. The purification of the enzyme was performed in the presence of (E)‐2‐methyl‐2‐butenoate as substrate to keep the enzyme in the oxidized state and under anaerobic conditions. The purification procedure included an ammonium sulphate precipitation, chromatography on DEAE‐Sepharose CL‐6B, hydroxylapatite and Sepharose CL‐6B. The enzyme reduces different α,β‐unsaturated carboxylate anions such as (E)‐2‐butenoate, (E)‐2‐methyl‐2‐butenoate, (E)‐cinnamate and probably many others in a NADH‐dependent reaction to the saturated carboxylate anions. Fumarate, 3‐phenyl‐2‐propinate, 2‐enoyl‐methyl and CoA esters proved not to be substrates for the purified reductase. NADPH does not act as an electron donor. The enzyme was shown to have a molecular weight of about 450000 by gel chromatography. It consists of subunits with a molecular weight of 78000. Per subunit about 1 FAD, 3.5–3.8 atoms of iron and 4.0 labile sulphur atoms have been found indicating a conjugated iron‐sulphur flavoprotein. Copper could not be detected. The isoelectric point was 8.4. As shown by absorption spectroscopy the enzyme can be reduced by NADH and reoxidized with dichloroindophenol, hexacyanoferrate III, oxygen and substrates. Addition of 8 mol p‐hydroxymercuribenzoate to 1 mol subunit completely destroyed the activity of the reductase. So far no physiological role of the enzyme is known.

Maleylacetate reductase of Pseudomonas sp. strain B13: specificity of substrate conversion and halide elimination

“…In an analogy to a reaction mechanism suggested for an enzyme (EC 1.3.1.31) reducing the carbon-carbon double bond of ␣,␤-unsaturated carboxylate anions of the methylbutenoate or cinnamate type (16), which also eliminate halide from the double bond (14), we propose that the following mechanism could play a role in the enzyme-catalyzed dehalogenation of maleylacetates when the substrate is used in the form of hydroxymuconate (Fig. 5A [the numbering of carbon has been made uniform for hydroxymuconate and maleylacetate to facilitate the discussion]).…”

On the formation of 3‐phenylpropionate and the different stereo‐chemical course of the reduction of cinnamate by Clostridium sporogenes and Peptostreptococcus anaerobius

“…We described enoate reductase a conjugated Fe-S flavoprotein which reduces nonactivated (II, P-unsaturated carboxylates in a NADH-dependent reaction [6]. The source was C. La 1 a non-proteolytic clostridium grown on (E)-2-butenoate.…”

The genome ofClostridium kluyveri, a strict anaerobe with unique metabolic features

“…It was shown that cell extracts of C. kluyveri catalyze the stereospecific reduction of a wide variety of ␣,␤-unsaturated fatty acids with H 2 . In the genome of C. kluyveri nine CDS for enoate reductases are found, only one of which (CKL 0743) has been characterized (45,46). The physiological function of the enoate reductases is not known with certainty.…”