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The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH2H4folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs. [ABSTRACT FROM AUTHOR]

Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O2 to H2O2, using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH2H4folate competitively inhibits the oxidase activity, which indicates that CH2H4folate and O2 compete for the same reduced and dUMP-activated enzymatic complex (FDTS–FADH2–NADP+–dUMP). These studies imply that the binding of CH2H4folate precedes NADP+ release during FDTS activity. The inhibition constant of CH2H4folate towards the oxidase activity was determined to be rather small (2 μm), which indicates a tight binding of CH2H4folate to the FDTS–FADH2–NADP+–dUMP complex. [ABSTRACT FROM AUTHOR]

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2′-deoxyuridine-5′-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2′-deoxyuridine-5′-monophosphate. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an 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 pathogens. 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 (that is, 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. These observations indicate a very different chemical cascade than that of classical thymidylate synthases 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. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs. [ABSTRACT FROM AUTHOR]