Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase

Abstract

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the major methyl donor for the conversion of homocysteine to methionine. Two common polymorphisms of the human enzyme have been identified: 677C>T, which leads to the substitution of Ala-222 by valine, and 1298A>C, which leads to the replacement of Glu-429 by alanine; the former polymorphism is the most frequent genetic cause of mild hyperhomocysteinemia, a risk factor for cardiovascular disease. By using a baculovirus expression system, recombinant human MTHFR has been expressed at high levels and purified to homogeneity in quantities suitable for biochemical characterization. The Glu429Ala protein has biochemical properties that are indistinguishable from the wild-type enzyme. The Ala222Val MTHFR, however, has an enhanced propensity to dissociate into monomers and to lose its FAD cofactor on dilution; the resulting loss of activity is slowed in the presence of methyltetrahydrofolate or adenosylmethionine. This biochemical phenotype is in good agreement with predictions made on the basis of studies comparing wild-type Escherichia coli MTHFR with a mutant, Ala177Val, homologous to the Ala222Val mutant human enzyme [Guenther, B. D., et al. (1999) Nat. Struct. Biol. 6, 359-365].

Figure 1

FAD dissociation after dilution of wild-type and mutant enzymes. Released FAD was detected by fluorometry. The enzyme solution was incubated at 37°C. (A) The change in fluorescence over time is shown for wild-type and Ala222Val mutant enzyme diluted to 50 nM. (B) The initial rate of release of FAD is plotted against enzyme concentration after dilution for the wild-type and mutant enzymes: wild type (●), Ala222Val (○), Glu429Ala (■), and Ala222Val Glu429Ala (□).

Figure 2

Protection of activity loss after dilution in the presence of CH₃-H₄folate. Enzyme was incubated with (6S)CH₃-H₄folate (Eprova) at 37°C. The residual enzyme activity after dilution was determined by CH₃-H₄folate:menadione oxidoreductase assay. (A) The time course of activity loss of Ala222Val mutant enzyme with (●) or without (○) 200 μM CH₃-H₄folate. (B) The rate constant for initial activity loss of wild-type (●), Ala222Val (○), Glu429Ala (■), and Ala222Val Glu429Ala (□) mutant enzymes is plotted against the concentration of CH₃-H₄folate present after dilution. The enzyme concentration after dilution is 50 nM.

Figure 3

Effect of AdoMet and AdoHcy on the initial rate of FAD release after dilution. Conditions are similar to those in Fig. 2. The initial rate of release of FAD is plotted against AdoMet concentration after dilution for the wild-type and mutant enzymes: wild type (●), Glu429Ala (■), and Ala222Val (○). The solid lines are fitted to data obtained in the absence of AdoHcy, and the dashed lines are fitted to data obtained in the presence of 100 μM AdoHcy and the indicated concentrations of AdoMet. Data for Ala22Val and for the double Ala222Val Glu429Ala mutant were indistinguishable (data not shown).

Figure 4

Protection against heat inactivation by (6-R, S)CH₃-H₄folate with or without added FAD. Purified wild-type, Ala222Val (A222V), Glu429Ala (E429A), and Ala222Val Glu429Ala (A>V & E>A) mutant MTHFRs were preincubated at 46°C in the presence of 0, 120, 240, or 360 μM (6-RS)CH₃-H₄folate without (A) or with (B) 10 μM FAD. After cooling to 37°C, the residual enzyme activity was determined by CH₃-H₄folate:menadione oxidoreductase assay. The values are expressed as mean ± SD of data obtained from three experiments. Statistically significantly differences (P < 0.03) from the activity of the same variant in the absence of CH₃-H₄folate are indicated by an asterisk. The different concentrations of CH₃-H₄folate are represented by the fill pattern of the bars: 0, black; 120, hatched; 240, white; and 360 μM, checkered.

Figure 5

Proposed mechanism for FAD dissociation. Each subunit consists of a catalytic (yellow) and a regulatory (white) domain, connected by a linker. The homodimer dissociates into monomers in a rapid and reversible step that is followed by rate-limiting FAD release.