Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

J Bacteriol. 1999 Feb;181(3):718-25. doi: 10.1128/JB.181.3.718-725.1999.


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 methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax 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 Km. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 5,10-Methylenetetrahydrofolate Reductase (FADH2)*
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins*
  • Flavin-Adenine Dinucleotide / analysis
  • Histidine
  • Kinetics
  • Macromolecular Substances
  • Methylenetetrahydrofolate Dehydrogenase (NAD+)
  • Molecular Weight
  • Oxidoreductases / genetics
  • Oxidoreductases / isolation & purification*
  • Oxidoreductases / metabolism*
  • Polymerase Chain Reaction
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • Escherichia coli Proteins
  • Macromolecular Substances
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide
  • Histidine
  • Oxidoreductases
  • Methylenetetrahydrofolate Dehydrogenase (NAD+)
  • 5,10-Methylenetetrahydrofolate Reductase (FADH2)
  • metf protein, E coli