Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: Roles for Aspartate 120 and Glutamate 28

The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHF R) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH2-H(4)folat e) to 5-methyltetrahydrofolate (CH3-H(4)folate). The X-ray crystal structur e of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis. Here, we have focused on two conse rved residues, Asp 120 and Glu 28, The presence of an acidic residue (Asp 1 20) near the N1-C2=O position of the flavin distinguishes MTHFR from all ot her known flavin oxidoreductases and suggests an important function for thi s residue in modulating the flavin reactivity. Modeling of the CH3-H(4)fola te product into the enzyme active site also suggests roles for Asp 120 in b inding of folate and in electrostatic stabilization of the putative 5-imini um cation intermediate during catalysis. In the NADH-menadione oxidoreducta se assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. C ompared to the wild-type enzyme, the mutant showed 150-fold decreased activ ity in the physiological NADH-CH2-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH2-H4folate, but the apparent Kd fo r CH2-H4folate was relatively unchanged. Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5 -iminium cation. By analogy to thymidylate synthase, which also uses CH2-H( 4)folate as a substrate, Glu 28 may serve directly or via water as a genera l acid catalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH2-H(4)f olate and was inactive in the physiological oxidoreductase reaction. The mu tant enzyme was able to bind CH3-H(4)folate, but reduction of the FAD cofac tor was not observed. In the NADH-menadione oxidoreductase assay, the mutan t demonstrated a 240-fold decrease in activity.