The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the NAD(P)H-dependent reduction of methylenetetrahydrofolate (CH2- H4folate) to methyltetrahydrofolate (CH3-H4folate). MTHFR, a key enzyme in folate metabolism, provides the substrate required for conversion of homocysteine to methionine. Patients with severe MTHFR deficiency have elevated levels of homocysteine and are at risk for neurological abnormalities and cardiovascular disease. Mild hyperhomocysteinemia is an indendent risk factor for cardiac disease, equivalent to that of hypercholoesterolemia or smoking. Elucidation of the catalytic mechanism of MTHFR, the goal of this research proposal, may help provide insights into methods of treatment for patients with MTHFR deficiency and hyperhomocysteinemia. The recently determined X-ray crystal structure of E. coli MTHFR, a model for the catalytic domain of the human enzyme, has provided a structural framework in which to examine the functional roles of certain amino acid residues at the flavin active site of the enzyme. This proposal focuses on two specific enzyme residues, Asp 120 and Glu 28. A conserved aspartic acid residue (Asp 120) above the N1 position of the flavin distinguishes MTHFR from all other known flavoproteins and suggests an important function for this residue in modulating the flavin reactivity. By analogy to the enzyme thymidylate synthase, which also uses CH2-H4folate as a substrate, a conserved glutamic acid residue (Glu 28) in MTHFR appears nicely positioned for activation of the CH2-H4folate substrate for catalysis. These studies may provide insights into key mechanistic issues in both flavin and folate chemistry.
