Figure 1.
MTHFR non-covalently binds FAD as an essential cofactor. NAD(P)H and CH2-H4folate are physiological substrates. NAD(P)H reduces FAD, then the reduced FAD reduces CH2-H4folate. CH3-H4folate can reduce the oxidized FAD. The reduced FAD can be oxidized by menadione as an electron acceptor, which is routinely used for in vitro assays.
Figure 2.
SDS-PAGE, Molecular Mass and anaerobic titration of Thermus MTHFR.
A. Purity of Thermus MTHFR (tMTHFR) with or without a His-tag was analyzed by 12% polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained by Quick-CBB (Wako Pure Chemical, Osaka, Japan). The molecular markers were obtained from Bio-Rad. BPB, bromophenol blue. B. Measurement of the native molecular mass of the as-purified MTHFR by size exclusion column chromatography using the multi-angle light scattering detector. The red dots show the molecular weight at each point in the chromatogram, while the solid line shows the elution profile detected by the refractive index detector. C. Anaerobic titration of the as-purified MTHFR with NADH. Fifteen nmol of enzyme (based on FAD content) was prepared in the anaerobic cuvette, then titrated with NADH. The initial spectrum is shown in the red line, and the final one is blue. (inset) Changes in absorbance at 450 nm. D. Anaerobic titration of the FAD-replete MTHFR with (6RS)- 5-CH3-H4folate. Fifteen nmol of enzyme was used. The initial spectrum is shown in the red line. The final spectrum, which is shown in blue, is resemble to the spectrum of the fully reduced FAD, indicating that (6S) - 5-CH3-H4folate could reduce all FAD to the fully reduced form of FAD. (inset) Changes in absorbance at 450 nm.
Figure 3.
Catalytic properties of Thermus MTHFR.
A. NADH:menadione oxidoreductase activity of the as-purified (open circle) and the FAD-replete MTHFR (filled circle). Enzyme activities were determined with varying amounts of NADH. In this comparison, the same amounts of FAD bound subunits ([Et]) were used. (inset) Comparison of the enzyme activities using NADH and NADPH. NADH is a better substrate than NADPH for Thermus MTHFR. B. Temperature dependence of NADH: CH2-H4folate oxidoreductase activity using the as-purified MTHFR. Consumption of NADH was monitored for 1 min. Changes in absorbance measured without enzyme are expressed as “-Enz”. C. NADH: CH2-H4folate oxidoreductase activity of the as-purified (open circle) and the FAD-replete MTHFR (filled circle). The same amount of [Et] was used for assays of the as-purified and replete enzymes at 50°C. D. Substrate inhibition in CH3-H4folate:menadione oxidoreductase activity. The as-purified MTHFR was used for the assay and the activities were determined at various concentrations of (6S)-5-CH3-H4folate (up to 104 µM). The enzyme product, CH2-H4folate, was determined after conversion to CH+ = H4folate.
Table 1.
Steady-state kinetic parameter of Thermus MTHFR1.2.
Table 2.
Data collection and refinement statistics.
Figure 4.
Thermostability of Thermus MTHFR.
Thermostability of the as-purified (open symbols with dotted lines) and the FAD-replete MTHFR (filled symbols with solid lines). Diluted enzymes ([Et] = 2 µM) were incubated at 70°C (circle), 80°C (triangle), and 90°C (square) for the indicated times, then put on ice. The symbols of grey-colored triangles and diamonds with dashed lines represent the FAD-replete MTHFR with 10 µM FAD at 80 and 90°C, respectively. Enzyme activities were measured by NADH:menadione oxidoreductase assay.
Figure 5.
The dimers of the as-purified and the FAD-replete Thermus MTHFR.
A. The structure of the crystal of the as-purified MTHFR is shown in ribbon mode. The yellow colored subunit is the holo-subunit, which contains FAD drawn in stick mode. The blue colored subunit represent the apo-subunit that lacks the FAD cofactor in the active site. B. Ribbon drawing of the fold of the FAD-replete MTHFR. The dimer contains one FAD molecule in each subunit. FAD found in the subunit is drawn in stick mode along with the electron density from an omit map (|Fo|-|Fc| map, 3.0σ) computed after refinement.
Figure 6.
Comparison of quaternary structures of E. coli and Thermus MTHFRs.
A. A tetramer of E. coli MTHFR, viewed down the local two-fold axis, is drawn using the PDB file (1ZPT) as determined by Pejchal et. al. (Biochemistry (2006) 45, 4808-4818). B. A dimer of FAD-replete Thermus MTHFR is shown overlaid on E. coli MTHFR (transparent). Superimposed subunits are colored in yellow for both MTHFRs. It is clear that none of subunits of E. coli MTHFR can overlap the blue-colored subunit of the Thermus enzyme.
Figure 7.
Comparison with the apo- and holo-subunits of the as-purified enzyme.
A. Ribbon drawing of the center of the β8α8 barrel (the apo-subunit) is shown. Five amino acid residues (His77, Arg107, Gly108, Asp109, and Pro110) are shown in stick mode with a 2|Fo|-|Fc| map (1.0 σ). His77 interacts with Asp109. B. The active center of the holo-subunit is drawn in ribbon mode but five amino acid residues (His77, Arg107-Pro110) are shown in stick mode. FAD is shown in stick mode with an omit map (4.0 σ). The position of Asp109 is markedly changed by binding of FAD. C. A stereo view showing superimposition of the apo- and holo-subunits of the as-purified MTHFR dimer. Protein folding is drawn in ribbon mode. The FAD cofactor is shown in stick mode. The color scheme is the same as in the Figure 5A.
Figure 8.
Superimposition of the holo-subunits of the FAD-replete MTHFR.
Structures of the active sites of both holo-subunits of the dimer are compared. Positions of Glu18 and Asp109, which are homologous to catalytically important amino acid residues in the E. coli enzyme, are especially focused. Distances (Å) between atoms are also shown in brown and blue for the yellow and blue subunits in Figure 5B, respectively. Distances from O4 of ribityl chain of FAD to Oε of Asp109 were 2.58 and 2.71 Å for the yellow and blue subunits, respectively.
Figure 9.
Possible models for mammalian MTHFR dimer.
For a schematic model of mammalian MTHFR, catalytic and regulatory domains are shown in yellow with FAD and white, respectively. Using E. coli MTHFR as a model, mammalian MTHFR can be illustrated as in A, because the tetramer of E. coli MTHFR contains the local two-fold axis in the center of the tetramer (Figure 6A). Thermus MTHFR structure predicts the different model shown in B, in which the catalytic domains interact ‘side-by-side’.