title: Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.
abstract: 2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.
authors.affiliation: Department of Biotechnology, University of Tokyo, Japan.
authors.forename: K S
authors.initials: KS
authors.lastname: Yoon
authors.forename: M
authors.initials: M
authors.lastname: Ishii
authors.forename: Y
authors.initials: Y
authors.lastname: Igarashi
authors.forename: T
authors.initials: T
authors.lastname: Kodama
mesh_terms: D000595:Amino Acid Sequence; D001420:Bacteria, Aerobic / Q000201:enzymology* / Q000254:growth & development; D005288:Ferredoxins / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005288:Ferredoxins; D007656:Ketoglutaric Acids; D010088:Oxidoreductases