Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6.
Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an alphabetagammadelta-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80 degrees C. The pH optimum was 7.6-7.8. The apparent Km values for pyruvate and coenzyme A at 70 degrees C were 3.45 mM and 54 microM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t50%) at 70 degrees C was approximately 8 h.
Yoon
K S
KS
Department of Biotechnology, University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113, Japan.
Ishii
M
M
Kodama
T
T
Igarashi
Y
Y
D000595:Amino Acid Sequence; D001420:Bacteria, Aerobic / Q000201:enzymology* / Q000235:genetics; D004579:Electron Transport; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D050743:Pyruvate Synthase; D013379:Substrate Specificity; D013696:Temperature
D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase