Thesis (Ph. D.)--University of Iowa, 2007. / Thesis supervisor: Amnon Kohen. Includes bibliographical references (leaves 108-114).

Background: Significant interest in homocysteine exists due to its established role in embryogenesis, cardiovascular disease and neurotoxicity. This research investigates total plasma homocysteine in the context of carcinogenesis among healthy individuals aged 20 to 50 years. It is hypothesized that during this timeframe, elevated total plasma homocysteine (tHcy) concentration implicates a breakdown of the methionine-homocyteine biosynthesis pathway, in which folate deficiency, oxidative stress and altered DNA methylation capacity are potential consequences relevant to cancer etiology. Purpose: The overall purpose of this research is to identify novel genetic predispositions to a biomarker of cancer risk (tHcy). Interactions with dietary and genetic factors that act on this pathway are explored. Methods: The study population consisted of 284 healthy male and female volunteers recruited in Kingston, Ontario and Halifax, Nova Scotia between 2006 and 2008. Specifically, polymorphisms under consideration included: i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and ii) the GC single nucleotide polymorphism (G/C SNP) both found on the 5’untranslated region (UTR) of the TS gene, and iii) the 6 base pair deletion at base pair 1494 (TS1494del6) found on the 3’UTR. TS polymorphisms were categorized based on either 5’ or 3’ location and were dichotomized to either high or low TS expression. Gene-gene interactions between polymorphisms in TS and methylenetetrahydrofolate reductase (MTHFR C677T) on tHcy concentration were also analyzed. In addition, gene-diet interactions between serum folate and vitamin B12 status were examined. Results: Mean tHcy concentration for this study population was 8.65 µmol/L (standard deviation=1.96 µmol/L). After adjustment for confounders, higher mean tHcy levels of 0.48 μmol/L and 0.46 μmol/L were observed for the main effects of 5’polymorphisms (5’High) (p=0.04) and 3’polymorphism (3’High) (p=0.05), respectively. The largest difference in mean tHcy concentration was observed for the joint effects of TS polymorphisms (µ=0.74 μmol/L, p=0.11). Gene-gene interaction was observed between TS and MTHFR polymorphisms on tHcy concentrations (p<0.01). Conclusions: The findings of this research provide evidence of an association between TS polymorphisms and tHcy concentrations. These results suggest that TS polymorphisms, independent of dietary factors, may lead to elevated tHcy levels and potentially contribute to cancer development. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2008-08-21 16:05:02.797

No description available.

All organisms must maintain an adequate level of thymidylate, which gets phosphorylated twice and then utilized by DNA polymerases for DNA replication that must precede cell division. Most organisms rely on classical thymidylate synthase (TSase) for this function. However, a subset of microorganisms – including a number of notable, widespread human pathogens – relies on an enzyme with a distinct structure and catalytic strategy. This enzyme is termed flavin-dependent thymidylate synthase (FDTS), as the flavin is required for thymidylate production. Because of this considerable orthogonality between FDTS and classical TSase, FDTS serves as a promising target for new therapeutics – one that could have only mild adverse effects on the host organism. FDTS catalyzes the reductive methylation of uridylate (2′-deoxyuridine-5′-monophosphate; dUMP) to yield thymidylate (2′-deoxythymidine-5′-monophosphate; dTMP). The methylene originally resides on CH2H4folate and is eventually transferred to the nucleotide. This methylene’s route to dUMP is unique in enzymology, and our experiments described herein strive to gain an understanding of the molecular details of its transfer. Compounds that mimic intermediates and transition states along this path are likely to bind FDTS tightly and could be leads for drugs, and our new insights could facilitate this. After methylene transfer is complete, a hydride transfer from flavin to the nucleotide occurs. We utilized rapid quench flow techniques in heavy water to follow the hydrogen transfers in FDTS; solvent isotope effects were measured and analyzed, furnishing evidence that the hydride transfer contributes to rate limitation. Reconstitution of the enzyme with unnatural flavins both reinforced these conclusions and suggested new hypotheses and experiments.

Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA replication. The only known enzyme performing de novo dTMP synthesis is thymidylate synthase (TS). The goals of this thesis were to provide biochemical evidence for the existence of TS in chlamydiae, to investigate the mechanism by which the parasite obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts dUMP into dTXP. In vitro experiments conclusively establish these findings by the demonstration of TS activity in extracts prepared from host-free chlamydial reticulate bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial strains can synthesize folates de novo; however strains vary significantly in their ability to transport preformed folates from the host cell. A C. trachomatis gene which is capable of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned. Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open reading frame shows absence of sequence homology to known TS. Unique in vitro characteristics shared by the enzyme activities from both chlamydial extract and recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.

Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA replication. The only known enzyme performing de novo dTMP synthesis is thymidylate synthase (TS). The goals of this thesis were to provide biochemical evidence for the existence of TS in chlamydiae, to investigate the mechanism by which the parasite obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts dUMP into dTXP. In vitro experiments conclusively establish these findings by the demonstration of TS activity in extracts prepared from host-free chlamydial reticulate bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial strains can synthesize folates de novo; however strains vary significantly in their ability to transport preformed folates from the host cell. A C. trachomatis gene which is capable of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned. Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open reading frame shows absence of sequence homology to known TS. Unique in vitro characteristics shared by the enzyme activities from both chlamydial extract and recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.

Thesis (Ph. D.)--Cornell University, August, 1999. / Vita. Includes bibliographical references (leaves 141-168).

Thymidylate synthase (TS), encoded by the ThyA gene, is essential for the growth and survival of Mycobacterium tuberculosis and therefore is a potential drug target. Thymidylate synthase binds both a substrate, 2'-deoxyuridine-5'monophosphate (dUMP) as well as a cofactor, (6R,S)-5,10-methylenetetrahydrofolate (mTHF), providing the ability to inhibit a single target by two separate classes of molecules. 5'-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is a very tight binding mechanism based inhibitor, shown to have a Ki of 2nM for Mtb TS. Pemetrexed and Raltitrexed are both anti-folates, targeting the cofactor binding site of thymidylate synthase. The x-ray crystal structures of Mycobacterium tuberculosis thymidylate synthase were solved in the binary complexes ThyA-dUMP and ThyA-FdUMP at 2.5 A and 2.4 A resolutions, respectively. The ternary complex, ThyA-dUMP-Pemetrexed was solved to a resolution of 1.7 A. The enzyme is comprised of 8 alpha-helices as well as 23% of the protein formed by beta-sheets, including the dimer interface which is a beta-sandwich. Examination of the dUMP binding site allowed the identification of key conserved residues that play a role in ligand binding and catalysis. Comparison of the dUMP-Pemetrexed ternary complex with that of the human crystal structure shows two fewer interactions in the Mtb enzyme. One is due to the replacement of a Met with a Val which doesn't allow hydrophobic interactions with the ring system of Pemetrexed, and the other is the replacement of an Asn with a Trp, depriving the Mtb protein of a hydrogen bond at the N7 of the pyrrolo ring. A spectrophotometric assay that monitored DHF formation was used to determine the inhibition of Pemetrexed and Raltitrexed on Mtb TS. Both were verified as noncompetitive inhibitors, and Pemetrexed was found to have an IC50 of 17muM and a Ki of 16.8muM, while Raltitrexed had an IC50 of 3.5muM and a Ki of 3.2muM.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 141-154).

Antibiotic resistance represents a real threat in the modern world. The problem of resistance is brought about by the fast evolution of bacteria, accelerated by misuse and over-prescription of antibiotics and compounded by the decline in the discovery and development of new classes of antibiotics. Consequently, new targets for antibiotics are in high demand. Flavin-dependent thymidylate synthase (FDTS), which is not present in humans and is responsible for the biosynthesis of a DNA building block in several human pathogens (e.g., M. tuberculosis, B. anthracis, H. pylori), is one such novel target. FDTS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to produce 2'-deoxythymidine-5'-monophosphate (dTMP), with N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄fol) serving as the carbon source and a nicotinamide cofactor as the electron source. No efficient inhibitors of FDTS are known, despite high-throughput screening attempts to find them. Intermediate and transition-state mimics are likely to bind the enzyme with greater affinity and hence have a better chance at inhibiting FDTS. Therefore, the understanding of the chemical mechanism of FDTS is critical to the informed design of compounds capable of disrupting its function in bacteria. We utilized various techniques, including chemical trapping of reaction intermediates, substrate isotope exchange and stopped-flow, to investigate the FDTS mechanism and determine what sets it apart from other pyrimidine methylases. We found that at least two different intermediates kinetically accumulate in the FDTS-catalyzed reaction. Both of these intermediates are trapped in acid in the form of 5-hydroxymethyl-dUMP, which has never been isolated in other uracil-methylating enzymes. Under basic conditions, however, the earlier intermediate is converted to a species with an unusual flavin-derived adduct, while the later intermediate is converted to dTMP product. Our experiments also suggest that dUMP is activated for the reaction by the reduced flavin - a substrate activation mechanism distinct from the one employed by the classical pyrimidine-methylating enzymes.