Structure of enoate reductase from a Clostridium tyrobutyricum (C. spec. La1).
Enoate reductase from Clostridium tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of about 90% pure enzyme in less than 10 h. A purity greater than 98% could be obtained by additional chromatography on Sephacryl S-300. The enzyme sediments in the analytical ultracentrifuge as a single, symmetrical boundary with a velocity of S(0)20,w = 24.9 S. Equilibrium ultracentrifugation yielded a molecular mass of 940 000 +/- 20 000 Da. The enzyme contains one type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit molecular mass of about 73 000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, iron and labile sulfur, the enzyme purified by the new method showed approximately 0.7 mol of FMN per mol of subunit. A dissociation product sedimenting at a velocity of S(0)20,w = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The molecular mass was 230 000 +/- 10 000 Da as shown by sedimentation equilibrium analysis. Thus it appears that the dissociation product is a trimer of the 73 000-Da subunit. The formation of the 10-S fragment by dissociation of the native enzyme is accompanied by the loss of most of the FMN, whereas the FAD content is not changed. The fragment catalysed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductase activity with NADH or methylviologen as cosubstrate was low. Electron micrographs of negatively stained enoate reductase show trigonal symmetry. The data suggest that enoate reductase is a dodecamer (tetramer of trimers) with tetrahedral symmetry.
D003013:Clostridium / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011487:Protein Conformation
D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019367:2-enoate reductase