Structure of Enoate Reductase from aClostridium tyrobutyricum (C. spec.La1)

Publisher Website
Google Scholar
Abstract
Enoate reductase from C. tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of .apprx. 90% pure enzyme in < 10 h. A purity >90% 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 .**GRAPHIC**. = 24.9 S. Equilibrium ultracentrifugation yielded a MW of 940,000 .+-. 20,000 Da (daltons). The enzyme contains 1 type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit MW of .apprx. 73,000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, FE and labile S, the enzyme purified by the new method showed .apprx. 0.7 mol of FMN/mol of subunit. A dissociation product sedimenting at a velocity of .**GRAPHIC**. = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The MW 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 catalyzed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductace 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.