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Abstract: Uracil methylation is essential for survival of organisms and passage of information from generation to generation with high fidelity. Two alternative uridyl methylation enzymes, flavin-dependent thymidylate synthase and folate/FAD-dependent RNA methyltransferase, have joined the long-known classical enzymes, thymidylate synthase and SAM-dependent RNA methyltransferase. These alternative enzymes differ significantly from their classical counterparts in structure, cofactor requirements and chemical mechanism. This review covers the available structural and mechanistic knowledge of the classical and alternative enzymes in biological uracil methylation, and offers a possibility of using inhibitors specifically aiming at microbial thymidylate production as antimicrobial drugs. [Copyright &y& Elsevier]

Abstract: For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. This reaction catalyzed by thyA or TYMS encoded thymidylate synthases is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. However, many microorganisms have recently been shown to produce a novel, flavin-dependent thymidylate synthase encoded by the thyX gene. Preliminary structural and mechanistic studies have shown substantial differences between these deoxyuridylate-methylating enzymes. Recently, both the chemical and kinetic mechanisms of FDTS have provided further insight into the distinctions between thyA and thyX encoded thymidylate synthases. Since FDTSs are found in several severe human pathogens their unusual mechanism offers a promising future for the development of antibiotic and antiviral drugs with little effect on human thymidylate biosynthesis. [Copyright &y& Elsevier]

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]