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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\n 2+<\/jats:sup>\n ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate\n \n \n \n \n \n M<\/mml:mi>\n \n g<\/mml:mi>\n A<\/mml:mi>\n \n 2<\/mml:mn>\n +<\/mml:mtext>\n <\/mml:mrow>\n <\/mml:msubsup>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n , while\n \n \n \n \n \n M<\/mml:mi>\n \n g<\/mml:mi>\n A<\/mml:mi>\n \n 2<\/mml:mn>\n +<\/mml:mtext>\n <\/mml:mrow>\n <\/mml:msubsup>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n together with Arg233 stabilize the inorganic phosphate (P\n \n i<\/jats:italic>\n <\/jats:sub>\n ); (ii) Asp168 and three water molecules coordinate\n \n \n \n \n \n M<\/mml:mi>\n \n g<\/mml:mi>\n B<\/mml:mi>\n \n 2<\/mml:mn>\n +<\/mml:mtext>\n <\/mml:mrow>\n <\/mml:msubsup>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n , and\n \n \n \n \n \n M<\/mml:mi>\n \n g<\/mml:mi>\n B<\/mml:mi>\n \n 2<\/mml:mn>\n +<\/mml:mtext>\n <\/mml:mrow>\n <\/mml:msubsup>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n further stabilizes P\n \n i<\/jats:italic>\n <\/jats:sub>\n by forming a hydrogen bond with two oxygens of P\n \n i<\/jats:italic>\n <\/jats:sub>\n ; (iii) Arg201 directly coordinates the P\n \n i<\/jats:italic>\n <\/jats:sub>\n ; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of\n \n \n \n \n \n M<\/mml:mi>\n \n g<\/mml:mi>\n A<\/mml:mi>\n \n 2<\/mml:mn>\n +<\/mml:mtext>\n <\/mml:mrow>\n <\/mml:msubsup>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n ,\n \n \n \n \n \n M<\/mml:mi>\n \n g<\/mml:mi>\n B<\/mml:mi>\n \n 2<\/mml:mn>\n +<\/mml:mtext>\n <\/mml:mrow>\n <\/mml:msubsup>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n and P\n \n i<\/jats:italic>\n <\/jats:sub>\n . Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected 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 important factors in determining the cofactor (NADP\n +<\/jats:sup>\n \/NAD\n +<\/jats:sup>\n ) 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 treatment.\n <\/jats:p>","DOI":"10.1371\/journal.pcbi.1010140","type":"journal-article","created":{"date-parts":[[2022,5,25]],"date-time":"2022-05-25T13:45:26Z","timestamp":1653486326000},"page":"e1010140","update-policy":"https:\/\/doi.org\/10.1371\/journal.pcbi.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase\/cyclohydrolase (MTHFD2)"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6552-0929","authenticated-orcid":true,"given":"Li Na","family":"Zhao","sequence":"first","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7247-7591","authenticated-orcid":true,"given":"Philipp","family":"Kaldis","sequence":"additional","affiliation":[]}],"member":"340","published-online":{"date-parts":[[2022,5,25]]},"reference":[{"key":"pcbi.1010140.ref001","doi-asserted-by":"crossref","first-page":"3128","DOI":"10.1038\/ncomms4128","article-title":"Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer","volume":"5","author":"R Nilsson","year":"2014","journal-title":"Nat Commun"},{"key":"pcbi.1010140.ref002","doi-asserted-by":"crossref","first-page":"1361","DOI":"10.1158\/1541-7786.MCR-15-0117","article-title":"Mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) overexpression is associated with tumor cell proliferation and is a novel target for drug development","volume":"13","author":"PM Tedeschi","year":"2015","journal-title":"Mol Cancer Res"},{"key":"pcbi.1010140.ref003","doi-asserted-by":"crossref","first-page":"14616","DOI":"10.1016\/S0021-9258(17)38612-X","article-title":"NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells","volume":"260","author":"NR Mejia","year":"1985","journal-title":"J Biol Chem"},{"key":"pcbi.1010140.ref004","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0006-291X(88)81040-4","article-title":"NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme","volume":"155","author":"NR Mejia","year":"1988","journal-title":"Biochem Biophys Res Commun"},{"key":"pcbi.1010140.ref005","doi-asserted-by":"crossref","first-page":"2339","DOI":"10.1038\/s41388-021-01695-8","article-title":"Therapeutic targeting of the mitochondrial one-carbon pathway: perspectives, pitfalls, and potential","volume":"40","author":"LN Zhao","year":"2021","journal-title":"Oncogene"},{"key":"pcbi.1010140.ref006","doi-asserted-by":"crossref","first-page":"e01021","DOI":"10.1016\/j.heliyon.2018.e01021","article-title":"Drug discovery of anticancer drugs targeting methylenetetrahydrofolate dehydrogenase 2","volume":"4","author":"A Asai","year":"2018","journal-title":"Heliyon"},{"key":"pcbi.1010140.ref007","first-page":"12","article-title":"Protein interaction and functional data indicate MTHFD2 involvement in RNA processing and translation. 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