Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima.
The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90 degrees C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113,000 and comprises four dissimilar subunits with M(r) values of approximately 43,000, 34,000, 23,000, and 13,000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent Km values at 80 degrees C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM, respectively, and the corresponding apparent Vm values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent Km and Vm values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 microM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90 degrees C, and the time for a 50% loss of activity (t50%) at 80 degrees C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25 degrees C of 70 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Blamey
J M
JM
Department of Biochemistry, University of Georgia, Athens 30602.
Adams
M W
MW
D000595:Amino Acid Sequence; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D050743:Pyruvate Synthase; D017386:Sequence Homology, Amino Acid
D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase