Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier.

Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N5-N10-methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity.

Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target.

In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

Chemical quenching and identification of intermediates in flavoenzyme-catalyzed reactions.

Chemical quenching offers a complementary approach to studying the mechanism of a flavoenzyme, supplementing the information learned from spectroscopic, structural, and computational methods. Generally, in a chemical quench experiment, an enzymatic turnover is quickly stopped at various stages with a chemical agent, and the individual reaction mixtures at each time point are analyzed for the reactants, products and any intermediates. The order by which bonds are made and broken in the reaction is indicated by the identities of the captured intermediates, and the rates of individual steps in the mechanism are determined from the amounts of various chemical species at different time points. This chapter outlines general considerations in selecting a chemical quencher of a particular enzyme-catalyzed reaction and methods for analyzing captured reaction intermediates, with a focus on flavoenzymes. The investigation of flavin-dependent thymidylate synthase is used as a case study to illustrate the concepts and workflow of quenching, isolating, and characterizing quencher-modified reaction intermediates and drawing mechanistic conclusions from the identities of these molecules.

An unprecedented mechanism of nucleotide methylation in organisms containing thyX.

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.