Fig. 2: Enzymatic characterization of S6MTHFR.
a Anaerobic titration of S6MTHFR with CH3-THF. In an anaerobic chamber, 19 nmol of S6MTHFR (oxidised FAD) was titrated with 25 nmol of CH3-THF to reach saturation. The initial spectrum is shown by the blue line, and the final spectrum is black. The arrow indicates the maximum absorption wavelength, 450 nm, of FAD. Inset: Changes in the amount of oxidised FAD (measured at 450 nm) with the addition of CH3-THF. Reactions were performed once. b HPLC chromatograms of reaction mixtures using 20 nM S6MTHFR and 200 μM CH3-THF in the presence of 400 μM menadione at 30 °C. The retention times of CH3-THF and CH2-THF were 1.44 and 2.12 min, respectively. c, d ESI–MS (positive-ion mode) spectra of CH3-THF and CH2-THF observed at the start and after 5 min of the reaction, respectively. m/z values for the protonated molecular ions [M + H]+ of CH3-THF and CH2-THF were 460 and 458, respectively. e Steady-state kinetic analysis of S6MTHFR. Optimal pH and temperature were determined. Reactions were carried out in four independent experiments (n  =  4). Data points marked with circles represent the means of the replicate measurements. Dots show individual data points. f, g Optimal pH and temperature were determined. Reactions were carried out in four independent experiments (n = 4). Data points marked with circles represent the means of the replicate measurements. Dots show individual data points.
