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

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofola te reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF; with six histidine resi dues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agaros e, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subun its, each of which contains a molecule of noncovalently bound flavin adenin e dinucleotide (FAB), No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate t o methyltetrahydrofolate, using NADH as the reductant, Kinetic parameters h ave been determined at 15 degrees C and pH 7.2 in a stopped-flow spectropho tometer; the K-m for NADH is 13 mu M, the K-m for CH2-H-4 folate is 0.8 mu M, and the turnover number under V-max conditions estimated for the reactio n is 1,800 mol of NADH oxidized min(-1) (mol of enzyme-bound FAD)(-1). NADP H also serves as a reductant, but exhibits a much higher K-m. MetF also cat alyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The pr operties of MetF from E, coli differ from those of the ferredoxin dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostri dium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes fr om eukaryotes.