Publications

• The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia.

  Guenther B.D., Sheppard C.A., Tran P., Rozen R., Matthews R.G., Ludwig M.L.

  Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (M ... >> More

  Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate. The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T). The X-ray analysis of E. coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a novel fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor. Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels. << Less

  Nat. Struct. Biol. 6:359-365(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients.

  Burda P., Schaefer A., Suormala T., Rummel T., Buerer C., Heuberger D., Frapolli M., Giunta C., Sokolova J., Vlaskova H., Kozich V., Koch H.G., Fowler B., Froese D.S., Baumgartner M.R.

  5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patient ... >> More

  5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations. << Less

  Hum. Mutat. 36:611-621(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of methylenetetrahydrofolate reductase from pig liver.

  Daubner S.C., Matthews R.G.

  Methylenetetrahydrofolate reductase from pig liver has been purified to homogeneity, as judged by several criteria: (i) a single band with a subunit molecular weight of 77,300 following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; (ii) a molecular weight determined ... >> More

  Methylenetetrahydrofolate reductase from pig liver has been purified to homogeneity, as judged by several criteria: (i) a single band with a subunit molecular weight of 77,300 following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; (ii) a molecular weight determined by amino acid analysis of 74,500 per flavin, in agreement with the subunit molecular weight; and (iii) constant specific activities in the peak fractions during the final chromatography step. The purified enzyme exhibits a typical flavoprotein absorption spectrum. Methylenetetrahydrofolate reductase is a minor constituent of pig liver, and to obtain homogeneous enzyme, a 32,000-fold purification must be accomplished. The preparation described herein attains such purification in 5 steps and with a 14% yield. The enzyme isolated in this fashion is active and stable, and contains a stoichiometric complement of FAD. The enzyme is reducible under anaerobic conditions by 5-deazaflavin/EDTA/light or by NADPH. Reduction of 1 mol of enzyme-bound FAD requires 1.1 mol of NADPH. The reduced enzyme can be reoxidized by (6-R)-methylenetetrahydrofolate, again with nearly 1:1 stoichiometry. Steady state kinetic measurements of the NADPH-methylenetetrahydrofolate oxidoreductase activity give parallel line double reciprocal plots. The turnover number per mol of enzyme-bound flavin is 1600/min under Vmax conditions. The spectrum of the enzyme-bound flavin is significantly perturbed by the binding of S-adenosylmethionine, a metabolite known to be an allosteric modulator of the enzyme. << Less

  J Biol Chem 257:140-145(1982) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mammalian methylenetetrahydrofolate reductase. Partial purification, properties, and inhibition by S-adenosylmethionine.

  Kutzbach C., Stokstad E.L.

  Biochim Biophys Acta 250:459-477(1971) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Sheppard C.A., Trimmer E.E., Matthews R.G.

  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 ... >> More

  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. << Less

  J. Bacteriol. 181:718-725(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.