Some structures of marine natural products

Finer-Moore, J.

doi:10.31274/rtd-180814-2405

Cited by 1 publication

(7 citation statements)

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“…To this date, the relevance of the folate/FAD-dependent tRNA methyltransferase methylating the uracil moiety of 2′-deoxyuridine-5′-monophosphate (dUMP). 53,54 There are two currently known classes of TSases that differ in structure, sequence, and cofactor requirements. 55 The homodimeric enzymes encoded by the thyA gene (the TYMS gene in humans) are referred to here as classical TSases.…”

Section: Discussionmentioning

confidence: 99%

“…Classical TSase enzymes use N 5 ,N 10 -methylenetetrahydrofolate (CH 2 H 4 folate) for both the one-carbon methylene and the reducing hydride to form the C7 methyl of the dTMP product ( Figure 2.1a). 54 The more recently discovered thyX-encoded proteins utilize a non-covalently-bound flavin adenine dinucleotide (FAD) prosthetic group to catalyze the redox chemistry and use TSase. 53,54 b) A mechanism proposed for FDTS where dUMP reduction occurs prior to methylene transfer.…”

Section: Discussionmentioning

confidence: 99%

“…54 The more recently discovered thyX-encoded proteins utilize a non-covalently-bound flavin adenine dinucleotide (FAD) prosthetic group to catalyze the redox chemistry and use TSase. 53,54 b) A mechanism proposed for FDTS where dUMP reduction occurs prior to methylene transfer. 56 c) An alternative mechanism proposed for FDTS where reduction happens after methylene transfer.…”

Section: Discussionmentioning

confidence: 99%

“…Thymidylate is essential for survival of all organisms, since without a pool of thymidylate for DNA biosynthesis cellular reproduction ceases. 1 In RNA, methylated uracils are found at a single site in tRNAs and two sites in the ribosome. 2 The 5-methyluridyl at position 54 in the T-loop of tRNAs in almost all living cells (designated as T54) plays a role in maintaining the tertiary structure of tRNA.…”

Section: Introductionmentioning

confidence: 99%

“…Such bifunctional classical TSase-DHFR enzymes are common in protozoa 25 and plants. 26 However, several observations have ruled out the possibility of FDTS being a bifunctional catalyst: (i) in studies with (R)-[6-3 H]-CH 2 H 4 folate, the tritium was retained by H 4 folate and not transferred to dTMP, 27 as with the classical TSase; 1,28 (ii) in contrast to classical TSase, reactions carried out in D 2 O produced deuterated dTMP, which points to proton exchange between the reduced flavin and the solvent prior to a hydride transfer from the flavin directly to the nucleotide; and (iii) when using tritiated NADPH to reduce the flavin, the tritium was found in water and not H 4 folate, 27 ruling out a flavin-dependent DHFR functionality of FDTS. Overall these findings support a mechanism in which NADPH reduces FAD to FADH 2 and the uracil moiety accepts a hydride from the FADH 2 to form dTMP.…”

Section: Introductionmentioning

confidence: 99%

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Flavin-dependent thymidylate synthase

Mishanina¹

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for teaching me electrochemistry, and Prof. M. Todd Washington for lending his quench-flow instrument and making me feel welcome in his lab space. The Department of Chemistry has been my home for the past six years, and I will miss everyone in it very much. Last but certainly not least, my thanks go out to everyone in Kansas (my American family) and my amazing mother, Elena Mishanina (my Tajikistan family). Without Pat Radford, Lanora Turner and Dr. Bill Gardner I would not be where I am today. In 2004, my mother watched her 16-year-old daughter leave for a foreign country literally halfway around the world away. We have been apart ever since. It is difficult for me, but I cannot even imagine how hard it is for her. I want to thank her for her sacrifices, support and faith in my abilities and judgment. vi ABSTRACT 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 N 5 ,N 10-methylene-5,6,7,8tetrahydrofolate (CH 2 H 4 fol) 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 flavina substrate vii activation mechanism distinct from the one employed by the classical pyrimidinemethylating enzymes. viii TABLE OF CONTENTS LIST OF FIGURES .

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Section: Discussionmentioning

confidence: 99%