title: An unprecedented mechanism of nucleotide methylation in organisms containing thyX.
abstract: In several human pathogens, thyX-encoded flavin-dependent thymidylate synthase (FDTS) catalyzes the last step in the biosynthesis of thymidylate, one of the four DNA nucleotides. ThyX is absent in humans, rendering FDTS an attractive antibiotic target; however, the lack of mechanistic understanding prohibits mechanism-based drug design. Here, we report trapping and characterization of two consecutive intermediates, which together with previous crystal structures indicate that the enzyme's reduced flavin relays a methylene from the folate carrier to the nucleotide acceptor. Furthermore, these results corroborate an unprecedented activation of the nucleotide that involves no covalent modification but only electrostatic polarization by the enzyme's active site. These findings indicate a mechanism that is very different from thymidylate biosynthesis in humans, underscoring the promise of FDTS as an antibiotic target.
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA. amnon-kohen@uiowa.edu.
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Mishanina
authors.affiliation: Nuclear Magnetic Resonance (NMR) Core Facility and Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Kalani
authors.initials: K
authors.lastname: Karunaratne
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Dibyendu
authors.initials: D
authors.lastname: Mondal
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Corcoran
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Choi
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D019175:DNA Methylation; D005415:Flavins / Q000737:chemistry; D005492:Folic Acid / Q000737:chemistry; D058972:Folic Acid Transporters / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D020124:Thermotoga maritima / Q000201:enzymology; D013938:Thymidine Monophosphate / Q000096:biosynthesis* / Q000737:chemistry; D013940:Thymidylate Synthase / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D058972:Folic Acid Transporters; D013938:Thymidine Monophosphate; D005492:Folic Acid; D013940:Thymidylate Synthase
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