JOURNAL OF BACTERIOLOGY, 0021-9193/99/$04.00+0 Feb. 1999, p. 718–725 Vol. 181, No. 3 Copyright © 1999, American Society for Microbiology. All Rights Reserved. Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from Escherichia coli CHRISTAL A. SHEPPARD, ELIZABETH E. TRIMMER, AND ROWENA G. MATTHEWS* Biophysics Research Division and Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Received 9 October 1998/Accepted 9 November 1998 A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydro- folate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the K m for NADH is 13 M, the K m for CH 2 -H 4 folate is 0.8 M, and the turnover number under V max conditions estimated for the reaction is 1,800 mol of NADH oxidized min 1 (mol of enzyme-bound FAD) 1 . NADPH also serves as a reductant, but exhibits a much higher K m . MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes. In Escherichia coli, methylenetetrahydrofolate reductase (MetF) catalyzes the reduction of 5,10-methylenetetrahydro- folate to 5-methyltetrahydrofolate. This reaction commits tet- rahydrofolate-bound one-carbon units to use in the methyl- ation of homocysteine to form methionine, the terminal step in methionine biosynthesis. Hatch et al. (14) first identified the enzyme activity in crude extracts of E. coli. NADH was shown to be more effective than NADPH as the source of reducing equivalents in relatively crude preparations (4). During puri- fication of methylenetetrahydrofolate reductase from cell ex- tracts, the ability of the enzyme to be reduced by NADH was lost, necessitating assay of the enzyme in the presence of NADH, an NADH-flavine adenine dinucleotide (FAD) oxi- doreductase, and FAD (17). Thus, it was believed that catalysis of the overall reaction shown in equation 1 required two en- zymes: a methylenetetrahydrofolate reductase that catalyzed transfer of reducing equivalents from reduced FAD to CH 2 - H 4 folate, and an NADH-FAD oxidoreductase that catalyzed transfer of reducing equivalents from NADH to FAD. NADH + CH 2 -H 4 folate 3 NAD + + CH 3 -H 4 folate (1) The metF gene specifies methylenetetrahydrofolate reductase. This gene is located in the metJBLF gene cluster, which was cloned and mapped by Zakin et al. (35). The metF gene was sequenced, and the gene product was shown to be a polypep- tide of 33 kDa (24). However, we are unaware of publications reporting further characterization of the E. coli enzyme. Methylenetetrahydrofolate reductase has previously been purified from porcine liver (7) and has been shown to contain noncovalently bound FAD and to use NADPH as a reductant. By using peptide sequences from the porcine enzyme to design oligomers, a clone for the human MTHFR gene was identified and sequenced, and the catalytic domain of the human enzyme was shown to exhibit extensive sequence similarity with MetF from E. coli (12). While two other bacterial methylenetetrahy- drofolate reductases have been purified and characterized, their sequences have not been reported and they appear to differ appreciably from the human and E. coli enzymes. The enzyme from Clostridium formicoaceticum (5) is an iron-sulfur flavoprotein that catalyzes reduction of CH 2 -H 4 folate with re- duced ferredoxin as an electron donor. Methylenetetrahydro- folate reductase from Peptostreptococcus productus more closely resembles the porcine and E. coli enzymes in that it lacks iron and catalyzes the reduction of methylenetetrahydro- folate with NADH as the electron donor (34). However, this enzyme appears to be associated with the cell membrane, in contrast to the E. coli and mammalian enzymes. In this paper, we report the construction of E. coli strains for overproduction of methylenetetrahydrofolate reductase and the purification and characterization of the enzyme. The E. coli enzyme serves as a useful model for the human enzyme, mu- tations of which have been implicated in hyperhomocysteine- mia in humans and in risk for the development of cardiovas- cular disease (10, 16) and neural tube defects (29). In work to be reported elsewhere, the X-ray structure of MetF from E. coli has been determined (13), and its availability will permit comparison with F 420 -dependent enzymes with similar func- tions in archaebacteria (22, 31). The ease of genetic manipu- lation of the E. coli metF gene will also permit further studies to define the functional properties of this important enzyme. We have purified the MetF protein to homogeneity and * Corresponding author. Mailing address: Biophysics Research Di- vision, The University of Michigan, Ann Arbor, MI 48109-1055. Phone: (734) 764-9459. FAX: (734) 764-3323. E-mail: rmatthew@umich.edu. 718 on January 18, 2017 by guest http://jb.asm.org/ Downloaded from