An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene

Abstract

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase (TS) to catalyze the methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP). All known thymidylate synthases (TSs) rely on an active site residue of the enzyme to activate dUMP1, 2. This functionality has been demonstrated for classical TSs, including human TS, and is instrumental in mechanism-based inhibition of these enzymes. Here we report the first example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3–5. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (i.e. a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2’-deoxythymidine-5’-monophosphate (dTMP). These observations indicate a very different chemical cascade than that of classical TSs or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Since several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

Folate binding site of flavin-dependent thymidylate synthase

“…2). (4,8). (B) A mechanism proposed for methylene transfer that involves an enzymatic arginine residue (16).…”

“…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).…”

“…Thymidylate synthases use N 5 , N 10 -methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate) to reductively methylate dUMP, producing dTMP. Two general classes of thymidylate synthases are known (4). The enzymes encoded by the thyA gene (TYMS in humans and other mammals) are well studied and are known as classic thymidylate synthases (TSase) (1,3).…”

“…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).…”

Genetic markers for Clostridium difficile lineages linked to hypervirulence

“…Its important role in microbial DNA replication and presence in several pathogenic bacteria makes it an attractive potential target for antibiotic drug development (Esra Ö nen et al Table 2). The two thymidylate synthetases use different mechanisms in the biosynthesis of dTMP (Koehn et al, 2009). In contrast to ThyX, which produces tetrahydrofolate (THF), ThyA forms dihydrofolate (DHF) and requires DHFR to produce THF (Koehn & Kohen, 2010).…”