Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

Kholodar, Svetlana A.; Kohen, Amnon

doi:10.1021/jacs.6b03826

Cited by 12 publications

(23 citation statements)

References 33 publications

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“…Scheme 1 shows the currently accepted mechanism 8 . The cofactor, mTHF, donates a methyl group to C5 of dUMP to produce covalent intermediate II (Scheme 1, step 1).…”

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confidence: 99%

A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer

2018

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In Escherichia coli thymidylate synthase (EcTS), rate-determining hydride transfer from the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate, to the intermediate 5-methylene-2’-deoxyuridine 5’-monophosphate occurs by hydrogen tunneling, requiring precise alignment of reactants and a closed binding cavity, sealed by the C-terminal carboxyl group. Mutations that destabilize the closed conformation of the binding cavity allow small molecules such as β-mercaptoethanol (β-ME) to enter the active site and compete with hydride for addition to the 5-methylene group of the intermediate. The C-terminal deletion mutant of EcTS produced the β-ME adduct in proportions that varied dramatically with cofactor concentration, from 50% at low cofactor concentrations to 0% at saturating cofactor conditions, suggesting communication between active sites. We report the 2.4Å X-ray structure of the C-terminal deletion mutant of E. coli TS in complex with substrate and a cofactor analog, CB3717. The structure is asymmetric, with reactants aligned in a manner consistent with hydride transfer in only one active site. In the second site, the CB3717 has shifted to a site where the normal cofactor would be unlikely to form 5-methylene-2’-deoxyuridine 5’-monophosphate, consistent with no formation of β-ME adduct. The structure shows how the binding of cofactor at one site triggers hydride transfer and borrows needed stabilization from substrate binding at the second site. It indicates pathways through the dimer interface that contribute to allostery relevant to half-sites reactivity.

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“…Scheme 1 shows the currently accepted mechanism 8 . The cofactor, mTHF, donates a methyl group to C5 of dUMP to produce covalent intermediate II (Scheme 1, step 1).…”

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confidence: 99%

A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer

2018

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“…Note that the peak observed with UV detection at 16.6 min in Fig. 1 A corresponds to [6- 3 H]H 4 F, which is in equilibrium with [6- 3 H]CH 2 H 4 F ( 15 ). Therefore, 3 H signal at 18–20 min in Fig.…”

Section: Resultsmentioning

confidence: 95%

“…Directed by the molecular mechanism outlined in Fig. 3 A and using KinTek Explorer, we globally fit the entire set of the rapid quench data and previously obtained steady-state and presteady-state kinetics of the reaction [including steady-state kinetics of externally added Int-B turnover ( 15 )] and found a solution consistent with the proposed pathway ( Fig. 2 A , global fit; SI Appendix has details).…”

Section: Resultsmentioning

confidence: 99%

“…In a critical test of previous QM/MM predictions ( 6 , 8 ), we recently showed that chemically synthesized Int-B is indeed a chemically and kinetically competent intermediate of the reaction ( 15 ). Interestingly, inspection of the kinetics of Int-B turnover by TSase suggested that its accumulation during the natural course of the reaction would be sufficient for its detection in a rapid quench experiment.…”

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confidence: 88%

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Parallel reaction pathways and noncovalent intermediates in thymidylate synthase revealed by experimental and computational tools

Kholodar

Ghosh

Świderek

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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SignificanceThymidylate synthase (TSase) is one of the prime anticancer targets, as it generates the sole de novo source of thymidylate, a unique precursor for DNA. Fluoropyrimidines and antifolates inhibiting TSase pathways are not only components of the most widely used therapeutic regimens but also highly toxic agents leading to acquired resistance in tumor cells. To provide fresh mechanistic insight to TSase-targeted drug design, we used a combination of experiments, kinetic modeling, and quantum mechanics/molecular mechanics (QM/MM) calculations to discover formation of unexpected noncovalent intermediates prevailing in the catalysis and overstabilized covalent species corresponding to a parallel reaction pathway. These findings challenge the traditional chemical path of this enzyme and offer drug targets.

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“…According to the reaction mechanism, the thiol of the conserved catalytic cysteine (Cys197 and Cys195, in Ef TS and hTS, respectively) attacks the carbon atom in position 6 (C6) on the nucleotide pyrimidine base. This leads to the formation of a covalent adduct (Scheme 1) [9,10]. The dUMP uracil carbon in position 5 (C5) is then activated to accept the methyl moiety (C11) and the hydride donated by mTHF.…”

Section: Introductionmentioning

confidence: 99%

Structural Comparison of Enterococcus faecalis and Human Thymidylate Synthase Complexes with the Substrate dUMP and Its Analogue FdUMP Provides Hints about Enzyme Conformational Variabilities

et al. 2019

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Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the half-site reactivity of Enterococcus faecalis TS and the mechanisms underlying the conformational changes occurring in the two enzymes. We also identify relevant differences in cofactor and inhibitor binding between Enterococcus faecalis and human TS that can guide the design of selective inhibitors against bacterial TSs.

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Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

Cited by 12 publications

References 33 publications

A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer

A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer

Parallel reaction pathways and noncovalent intermediates in thymidylate synthase revealed by experimental and computational tools

Structural Comparison of Enterococcus faecalis and Human Thymidylate Synthase Complexes with the Substrate dUMP and Its Analogue FdUMP Provides Hints about Enzyme Conformational Variabilities

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