title: Purification and characterization of an anabolic fumarate reductase from Methanobacterium thermoautotrophicum.
abstract: An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the cells of the archaebacterium Methanobacterium thermoautotrophicum. A major portion of the purification was performed inside an anaerobic chamber, employing reducing agents to maintain low redox potentials. The apparent molecular weight of the native enzyme is 78,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a minimal subunit molecular weight of about 20,000. Iodoacetamide (1 mM) and copper chloride (5 mM) caused significant loss in the enzyme activity. The optimum temperature for the enzymatic activity was 75 degrees C. The pH optimum was found to be 7.0. The fumarate reductase had an apparent Km of 0.20 mM for fumarate. Purified enzyme was colorless; spectroscopic studies indicated the absence of flavins as a cofactor. The spectral data, however, suggested the presence of an unknown cofactor tightly bound to the enzyme. Fumarate reductase is involved in the anabolic rather than the catabolic metabolism of M. thermoautotrophicum.
authors.affiliation: Environmental Sciences and Resources Program in Chemistry and Biology, Portland State University, Oregon 97207.
authors.forename: S S
authors.initials: SS
authors.lastname: Khandekar
authors.forename: L D
authors.initials: LD
authors.lastname: Eirich
mesh_terms: D000693:Anaerobiosis; D001105:Archaea / Q000201:enzymology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C020155:fumarate reductase (NADH)