Catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Abstract Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg 2+ ion, and (b) the substrate-bound form of MTHFD2. Using homology modeling and simulation studies, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate Mg A , while Mg A together with Arg233 stabilize the inorganic phosphate (P i ); (ii) Asp168 and three water molecules coordinate Mg B , and Mg B further stabilizes P i by forming a hydrogen bond with two oxygens of P i ; (iii) Arg201 directly coordinates the P i ; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of Mg A , Mg B and P i . Our computational study at the empirical valence bond level allowed us to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution coupled to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues Ser197/Arg233 are key factors in determining the cofactor (NADP + /NAD + ) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer therapy.