Methylenetetrahydrofolate reductase is a flavoprotein, which uses enzyme-bound FAD to catalyze the reduction of 5,10-methylenetetrahydrofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate). In the oxidative half-reaction, CH2-H4folate is proposed to accept a proton at the N10 position to open up the imidazolidine ring and form a 5-iminium cation intermediate, which undergoes reduction. Previous studies have shown that Glu28, located near N10, is essential for this half-reaction. Glu28, however, is believed to be negatively-charged. Thus, we have proposed that protonation is accomplished through a relay involving a triad of hydrogen-bonded residues: Ser26, His273, and Glu28. In order to elucidate the mechanism and importance of Ser26 in this proton shuttling, we have characterized two Ser26 mutant enzymes. We hypothesize that the Ser26Ala mutant enzyme will show reduced activity compared to the wild-type enzyme due to a lack of hydrogen bonding, and that the Ser26Thr mutant enzyme will have similar activity as the wild-type enzyme since it retains the hydrogen bonding. Our results indicate that Ser26Thr has similar activity as the wild-type enzyme, and that Ser26Ala has still-functional, but reduced activity.

Importance of Ser26 in the folate half-reaction of E. coli MTHFR

253rd National Meeting (2017)

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