Mechanistic Studies of a Flavin-Dependent Thymidylate Synthase

Abstract

The ThyA gene that encodes for thymidylate synthase (TS) is absent in the genomes of a large number of bacteria, including several human pathogens. Many of these bacteria also lack the genes for dihydrofolate reductase (DHFR) and thymidine kinase and are totally dependent on an alternative enzyme for thymidylate synthesis. Thy1 encodes flavin-dependent TS (FDTS, previously denoted as TSCP) and shares no sequence homology with classical TS genes. Mechanistic studies of a FDTS from Thermotoga maritima (TM0449) are presented here. Several isotopic labeling experiments reveal details of the catalyzed reaction, and a chemical mechanism that is consistent with the experimental data is proposed. The reaction proceeds via a ping-pong mechanism where nicotinamide binding and release precedes the oxidative half-reaction. The enzyme is primarily pro-R specific with regard to the nicotinamide (NADPH), the oxidation of which is the rate-limiting step of the whole catalytic cascade. An enzyme-bound flavin is reduced with an isotope effect of 25 (consistent with H-tunneling) and exchanges protons with the solvent prior to the reduction of an intermediate methylene. A quantitative assay was developed, and the kinetic parameters were measured. A significant NADPH substrate inhibition and large K M rationalized the slow activity reported for this enzyme in the past. These and other findings are compared with classical TS (ThyA) catalysis in terms of kinetic and molecular mechanisms. The differences between the FDTS proposed mechanism and that of the classical TS are striking and invoke the notion that mechanism-based drugs will selectively inhibit FDTS and will not have much effect on human (and other eukaryotes) TS. Since TS activity is essential to DNA replication, the unique mechanism of FDTS makes it an attractive target for antibiotic drug development.

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

“…In the alternative pathway, ThyX performs the transfer without oxidation of tetrahydrofolate, utilizing both dUMP and methylenetetrahydrofolate as reactants and FAD and NADPH in the redox phase of the reaction (1,10,17,22,23). Whereas ThyA requires a companion enzyme, dihydrofolate reductase, to regenerate tetrahydrofolate that has been oxidized in the transfer reaction, ThyX requires dUMP, NADPH, FAD, and methylenetetrahydrofolate (3).…”

“…In ThyX enzymes, the conversion of dUMP to dTMP involves two half reactions, oxidation and transfer of the methylene group from dUMP to form the dTMP final product (1). To test the ability of the ThyX mutants to execute these essential half reactions, two biochemical assays were performed.…”

Folate binding site of flavin-dependent thymidylate synthase

“…This observation led to the identification of alternative flavin-dependent thymidylate synthases (FDTSs), which are encoded by the thyX gene and have no sequence or structure homology to classic TSase enzymes (2,6,7). Furthermore, multiple studies have identified key differences in the molecular mechanism of catalysis between FDTSs and classic TSases (2,4,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). These differences, along with the fact that the thyX gene is present in many human pathogens (e.g., bacteria causing Anthrax, Tuberculosis, Typhus, and other diseases), renders these flavo-enzymes as potential targets for rational inhibitor design, possibly affording compounds that might be effective antimicrobial drugs (11,(22)(23)(24).…”

“…Although the reductive halfreaction is activated by dUMP the conversion of dUMP to dTMP occurs during the oxidative half-reaction (FADH 2 →FAD) (8,10,14,18,27). In contrast to classic TSase, where the cofactor CH 2 H 4 folate provides both the H − and methylene, in the FDTS reaction the H − is provided by the FADH 2 and CH 2 H 4 folate is used only as a source for the methylene moiety (18,20,28).…”

“…1A) from CH 2 H 4 folate to dUMP (4,8,10,(13)(14)(15)(18)(19)(20), or transfer of the methylene via an arginine residue (Arg174 in Thermatoga maritima FDTS; TmFDTS) before its delivery to dUMP (Fig. 1B) (16).…”