The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 pmol . min-' mg protein-' (37°C; pH 5.5). The apparent K , for NADH was near 10 pM. The apparent molecular mass of the enzyme was determined by gel filtration to be ~2 5 0 . 0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD' nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used as substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP+-dependent reductase of eucaryotes investigated so far.
The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophicaily grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein.The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 ,M, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric a2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to >95% in the soluble (supernatant) fraction. Sodium (10 FM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.Homoacetogenic bacteria are strictly anaerobic eubacteria that mediate the total synthesis of acetate from CO2 in their energy metabolism. The methyl group of acetate is formed from CO2 via formate and tetrahydrofolate-bound C-1 intermediates; synthesis of the carboxyl group proceeds via a bound carbon monoxide (for recent reviews on homoacetogens and their energy metabolism, see references 3, 7, 13, and 20). Most homoacetogenic bacteria are able to grow on H2 plus CO2 as energy sources. Therefore, reduction of CO2 to acetate must be coupled with a net formation of ATP. Studies with Acetobacterium woodii (4) led to the assumption that ATP synthesis is coupled to the formation of the methyl group of acetate via a chemiosmotic mechanism. For the homoacetogen Peptostreptococcus productus (9) and other homoacetogens (10, 21), evidence was presented that an electrochemical sodium gradient formed upon synthesis of the methyl group from formate could play an essential role in energy conservation. The sodium-dependent reaction in methyl group synthesis is not yet known. Therefore, we studied the enzymes mediating methyl formation. Of particular interest are the enzymes that catalyze reactions sufficiently exergonic to be possibly coupled to a sodium translocation across the cytoplasmic membrane. These reactions are, at least under standard conditions and with CO or H2 as an electron donor, the reduction of methylenetetrahydrofolate to methyltetrahydrofolate and the formation of methylenetetrahydrofolate from methenyltetrahydrofolate. The former reaction is the most exergonic step in m...
Washed cells of Peptostreptococcus productus (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/ 17%/ 33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis‐Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/1. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.
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