Trapping of the C5 Methylene Intermediate in Thymidylate Synthase

Barrett, Jeannie E.; Maltby, David; Santi, Daniel V.; Schultz, Peter G.

doi:10.1021/ja973210t

Cited by 35 publications

(46 citation statements)

References 15 publications

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“…This covalent ternary intermediate (Scheme 3A, between steps 3 and 4) has also been detected in quenching experiments with wild-type TSase 14 and by isolation on SDS-PAGE in reactions of E60A and E60L mutants of L. casei TSase with radiolabeled substrates. 15 The formation of the exocyclic methylene intermediate (Scheme 3A, between steps 4 and 5) was confirmed in experiments with a W82Y mutant of L. casei TSase, 16 which allowed premature release of H 4 folate from the active-site and subsequent chemical trapping of the intermediate with β-mercaptoethanol under steady-state conditions.…”

Section: Classical Thymidylate Synthase (Tsase Ec 21145) and Fmentioning

confidence: 90%

Mechanisms and inhibition of uracil methylating enzymes

Mishanina

Koehn

Kohen

2012

Bioorganic Chemistry

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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.

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Section: Classical Thymidylate Synthase (Tsase Ec 21145) and Fmentioning

confidence: 90%

Mechanisms and inhibition of uracil methylating enzymes

Mishanina

Koehn

Kohen

2012

Bioorganic Chemistry

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“…9 The existence of the exocyclic methylene intermediate (between steps 4 and 5) was supported by experiments with a W82Y mutant of Lc TSase, which allowed chemical trapping with β-mercaptoethanol under steady state conditions. 10 …”

Section: Introductionmentioning

confidence: 99%

Trapping of an Intermediate in the Reaction Catalyzed by Flavin-Dependent Thymidylate Synthase

et al. 2012

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Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. Recently it has been shown that FDTS catalysis does not rely on an enzymatic nucleophile and the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist the efforts to rationally design inhibitors as leads for future antibiotics.

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“…Although much is known about the mechanism of action of this unique enzyme, which is one of the most conserved in nature (1), it has not yet yielded all of its secrets. In particular, potential intermediates involved in the final hydride transfer step from tetrahydrofolate to methylene to yield the methyl group of dTMP have been proposed (3)(4)(5), but indisputable verification is still to be provided. Another area that is somewhat murky is how specific inactive mutants of Escherichia coli TS can exchange their subunits to yield a completely revitalized enzyme (6), in support of earlier evidence suggesting that TS is a half-the-sites of activity enzyme.…”

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confidence: 99%