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

• DOI: 10.1111/j.1432-1033.1979.tb13090.x
• Publication Date: 2005-03-03T20:01:03Z
• Authors: Wilhelm TISCHER; Johann BADER; Helmut SIMON
• Keywords: Carboxylate; Sepharose

2-Enoate-reductase, a previously unknown soluble enzyme is present in Clostridium kluyveri and another species growing on (E)-2-butenoate. From the latter reductase was purified 88-fold with an overall yield up to 74%. The pure as judged by polyacrylamide gel electrophoresis without sodium dodecyl sulphate well isoelectric focusing. purification of performed presence (E)-2-methyl-2-butenoate substrate keep oxidized state under anaerobic conditions. procedure included ammonium precipitation, chromatography DEAE-Sepharose CL-6B, hydroxylapatite Sepharose CL-6B. reduces different alpha,beta-unsaturated carboxylate anions such (E)-2-butenoate, (E)-2-methyl-2-butenoate, (E)-cinnamate probably many others NADH-dependent reaction saturated anions. Fumarate, 3-phenyl-2-propinate, 2-enoyl-methyl CoA esters proved not be substrates for reductase. NADPH does act electron donor. shown have molecular weight about 450,000 chromatography. It consists subunits 78,000. Per subunit 1 FAD, 3.5--3.8 atoms iron 4.0 labile sulphur been found indicating conjugated iron-sulphur flavo-protein. Copper could detected. point 8.4. As absorption spectroscopy can reduced NADH reoxidized dichloroindophenol, hexacyanoferrate III, oxygen substrates. Addition 8 mol p-hydroxymercuribenzoate completely destroyed activity So far no physiological role known.