JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1414-1419 Vol. 173, No. 4 0021-9193/91/041414-06$02.00/0 Copyright 0 1991, American Society for Microbiology Purification and Characterization of NADP+-Dependent 5,10-Methylenetetrahydrofolate Dehydrogenase from Peptostreptococcus productus Marburg GERT WOHLFARTH, GABRIELE GEERLIGS, AND GABRIELE DIEKERT* Institutfur Mikrobiologie, Universitat Stuttgart, Azenbergstrasse 18, D-7000 Stuttgart 1, Federal Republic of Germany Received 19 June 1990/Accepted 7 December 1990 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 membrane, thus pointing to its proposed role in energy that mediate the total synthesis of acetate from CO2 in their conservation. However, the methylenetetrahydrofolate re- energy metabolism. The methyl group of acetate is formed duction was not dependent on sodium (19). from CO2 via formate and tetrahydrofolate-bound C-1 inter- Here we report on the isolation and properties of the mediates; synthesis of the carboxyl group proceeds via a methylenetetrahydrofolate dehydrogenase to elucidate the bound carbon monoxide (for recent reviews on homoaceto- possible role of this enzyme in energy conservation of gens and their energy metabolism, see references 3, 7, 13, homoacetogenic bacteria. Our data can be interpreted as and 20). Most homoacetogenic bacteria are able to grow on excluding such a role in the homoacetogen P. productus. H2 plus CO2 as energy sources. Therefore, reduction of CO2 This conclusion is supported by an earlier observation, that to acetate must be coupled with a net formation of ATP. in A. woodii the formation of the methyl group from form- Studies with Acetobacterium woodii (4) led to the assump- aldehyde, which does not involve methylenetetrahydrofolate tion that ATP synthesis is coupled to the formation of the dehydrogenase, was also sodium dependent (10). methyl group of acetate via a chemiosmotic mechanism. For the homoacetogen Peptostreptococcus productus (9) and other homoacetogens (10, 21), evidence was presented that MATERIALS AND METHODS an electrochemical sodium gradient formed upon synthesis and Dowex 50 ofthe methyl group from formate could play an essential role Materials. Tetrahydrofolic acid, DNase II, reaction in WX 8, 100/200 mesh, were from Serva (Heidelberg, Federal in energy conservation. The sodium-dependent Republic of Germany). Formaldehyde, 2-mercaptoethanol methyl group synthesis is not yet known. Therefore, we Federal studied the enzymes mediating methyl formation. Of partic- and folic acid were from Fluka (Neu-Ulm, Republic reactions suffi- ofGermany). NAD(P)+ and flavin adenine dinucleotide were ular interest are the enzymes that catalyze from Boehringer (Mannheim, Federal Republic of Germa- ciently exergonic to be possibly coupled to a sodium trans- re- location across the cytoplasmic membrane. These reactions ny). The chromatography and column materials except and with CO or H2 as active yellow 86-agarose were purchased from Pharmacia are, at least under standard conditions (Uppsala, Sweden). Centricon 30 tubes were from Amicon an electron donor, the reduction of methylenetetrahydrofo- and all other late to methyltetrahydrofolate and the formation of methyl- (Danvers, Mass.). Reactive yellow 86-agarose enetetrahydrofolate from methenyltetrahydrofolate. The for- chemicals of the highest available purity were obtained from mer reaction is the most exergonic step in methyl group Sigma (Deisenhofen, Federal Republic of Germany). synthesis. Studies of the methylenetetrahydrofolate reduc- Analytical methods. Methylenetetrahydrofolate dehydro- thermoautotrophicum (11) genase was assayed routinely by photometric determination tase in vesicles of Clostridium of the reduction of NADP+ and formation of methenyltet- and of the same enzyme purified from P. productus (19) = enzyme could be associated with the rahydrofolate from methylenetetrahydrofole at 350 nm (e indicated that the 5.6 mM-' cm-1 for NADPH and 24.9 mM-1 cm-' for methenyltetrahydrofolate) and 37°C. The assays were con- ducted aerobically in cuvettes filled with 1 ml of anaerobi- * Corresponding author. cally prepared 100 mM potassium phosphate buffer (pH 5.5) 1414 VOL. 173, 1991 METHYLENE-FH4 DEHYDROGENASE FROM P. PRODUCTUS 1415 or 100 mM Tris-HCl (pH 7.5) containing 50 mM 2-mercap- near 5 ,M sodium. The reversibility of the methylenetet- toethanol, 0.5 mM tetrahydrofolic acid, 10 mM formalde- rahydrofolate dehydrogenase reaction was studied in the hyde, and 0.3 mM NADP+. Methylenetetrahydrofolate was same assay system in the presence and absence of added formed nonenzymatically from tetrahydrofolate and formal- sodium (5 mM). The following substrate concentrations were dehyde (18). Only 25% of the tetrahydrofolate initially added used: methylenetetrahydrofolate ('biologically active'), 50 were converted to biologically active methylenetetrahydro- ,uM; and NADP+, 100 ,uM. After the equilibrium of the folate, since (i) two stereoisomers of tetrahydrofolate are reaction was reached, 100 ,uM NADPH was added. The present in the commercially available substance and (ii) part decrease in indicated the reoxidation of the subsequent absorption tetrahydrofolate was in its oxidized state as dihydro- of NADPH with methenyltetrahydrofolate as the electron folate (19). The concentration of convertible methylenetet- acceptor. No reverse reaction was observed when the en- rahydrofolate was determined as described earlier (19) by zyme was irreversibly inactivated by heating the cuvette to limiting the tetrahydrofolate concentration in the methylene- 60°C for 10 min prior to the addition of NADPH. tetrahydrofolate reductase and dehydrogenase assays with Purification of the excesses methylenetetrahydrofolate dehydroge- of the other substrates (NADH and NADP+) and nase. P. productus (ATCC 43917) cells were grown on formaldehyde. The reaction was started by the addition of glucose medium as described earlier (8). The cells were protein. The kinetic properties of the enzyme were analyzed harvested by centrifugation in the late logarithmic growth by varying the concentrations of methylenetetrahydrofolate phase and stored frozen at -20°C. and NADP+ as the substrates and NADPH as the product of (i) 1. of crude extracts and ammonium the Step Preparation reaction (see Fig. 3 and 4). The methylenetetrahydrofo- sulfate treatment. Frozen cells were suspended in an equal late concentrations of convertible substrate are given in volume of 50 mM Tris-HCl (pH 7.5) containing 5 mM Results and Discussion. mercaptoethanol, 1 mM 1 mM To exclude the MgCl2, phenylmethylsulfonyl presence of methenyltetrahydrofolate cy- fluoride, and 0.2 mg of DNase II per ml. The suspension was clohydrolase activity in the purified enzyme, the cyclohy- passed twice through a French pressure cell at 100,000 kPa. drolase reaction was measured by the addition of crude The crude extract was centrifuged at 80,000 x g and 4°C for extracts to methylenetetrahydrofolate dehydrogenase as- 60 min. The supernatant was stirred on ice, and ice-cold 3.2 says, which had been previously incubated with purified M ammonium sulfate (pH 7.0) was added to a final concen- dehydrogenase until the methylenetetrahydrofolate had been tration of 1.9 M. After being stirred on ice for 10 min, the completely converted to methenyltetrahydrofolate, i.e., un- mixture was centrifuged at 20,000 x g for 10 min to remove til a constant absorption was reached. The decrease of the precipitated protein. absorption due to the conversion of methenyltetrahydrofo- (ii) 2. on The late to Step Chromatography phenyl-Sepharose. formyltetrahydrofolate indicated the presence of supernatant was passed through a phenyl-Sepharose Fast cyclohydrolase in the crude extracts (0.38 ,umol/min/mg of Flow column (1.0 by 15 cm) preequilibrated with 1.6 M protein at pH 7.5). ammonium sulfate in basic buffer (20 mM Tris-HCI [pH 7.5] Protein was determined with bovine serum albumin as the containng 0.5 mM dithiothreitol). The column was washed standard by the Coomassie blue method (Bio-Rad, Munich, with 1.6 and 1.1 M ammonium sulfate ml Federal