title: Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase.
abstract: Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.
authors.affiliation: Program in Chemical Biology, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Stull
authors.affiliation: Program in Chemical Biology, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Steffen M
authors.initials: SM
authors.lastname: Bernard
authors.affiliation: Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Aparna
authors.initials: A
authors.lastname: Sapra
authors.affiliation: Program in Chemical Biology, University of Michigan , Ann Arbor, Michigan 48109, United States.|Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.|Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
authors.affiliation: Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Erik R P
authors.initials: ER
authors.lastname: Zuiderweg
authors.affiliation: Program in Chemical Biology, University of Michigan , Ann Arbor, Michigan 48109, United States.|Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Palfey
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D005415:Flavins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D011522:Protons; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D005415:Flavins; D011522:Protons; D009249:NADP; D013940:Thymidylate Synthase