Mechanistic and structural basis for inhibition of thymidylate synthase ThyX

Basta, Tamara; Boum, Yap; Briffotaux, Julien; Becker, Hubert F.; Lamarre-Jouenne, Isabelle; Lambry, Jean−Christophe; Skouloubris, Stéphane; Liebl, Ursula; Graille, Marc; Tilbeurgh, Herman van; Myllykallio, Hannu

doi:10.1098/rsob.120120

Cited by 41 publications

(54 citation statements)

References 38 publications

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“…The inhibitors display in vivo potency against cisplatin‐resistant cancer cells, illustrating the potential of the TS inactive state as a therapeutic target . Other groups have demonstrated selective inhibition of ThyX, from the flavin‐dependent family of TS, by a molecule that binds at the interface between the individual monomers . There is, therefore, a potential to take advantage of conformational switching in the design of both active site and allosteric inhibitors of T. gondii TS–DHFR.…”

Section: Discussionsupporting

confidence: 52%

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

2013

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The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking bstrands at the interface, revealing a previously unknown allosteric target. The current study shows that these b-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

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Section: Discussionsupporting

confidence: 52%

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

2013

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“…We conjecture that the rapid large amplitude motions of the active site monitored in ThyX enzymes may be related to their capacity of interacting with multiple different aromatic and relatively rigid substrates and products. The method that we have explored here can be used to study the effect of other substrates and, in particular, inhibitors, including potential antimicrobial agents (18), on the dynamics of the active site of ThyX. More generally, our results emphasize the important role of enzyme dynamics in the interaction with the substrates.…”

Section: Discussionmentioning

confidence: 71%

“…ThyX shows no structural homology to thymidylate synthase ThyA, which is used in most eukaryotes (17). Because the ThyX pathway is used by a number of pathogenic bacteria and absent in humans, ThyX is considered a promising antimicrobial target (16,18); it catalyses carbon transfer from N 5 ,N 10 -methylene-5,6,7,8-tetrahydrofolate (MTHF or CH 2 H 4 folate) to deoxyuridine monophosphate (dUMP) using NADPH as a hydride donor and consequently has three substrates (dUMP, MTHF, NADPH) with the flavin adenine dinucleotide cofactor shuttling between the fully oxidized (FAD) and fully reduced (FADH 2 ) forms. dUMP binds in close interaction with the flavin group, displacing a nearby Tyr residue (19).…”

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confidence: 99%

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

Laptenok

Bouzhir-Sima

Lambry

et al. 2013

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

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In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.protein dynamics | flavoprotein | ultrafast fluorescence spectroscopy | quenching C onfigurational flexibility is essential for enzyme function during catalysis. Binding of one or more substrates, accommodation of the transition state where the actual reaction takes place, relaxation to the product state, and release of the product (s) is possible because different configurations of the enzyme are continuously sampled, by thermal or reaction-driven motions, on timescales ranging from femtoseconds to microseconds. The configurational space sampled in a certain time range will depend on the local protein flexibility/energy landscape and the temperature (1). During these configurational changes, distances between constituents of the enzyme complex change. It has been recognized that the fastest (femtosecond to picosecond) localized motions exist alongside the slower motions that occur on the (typically millisecond) timescale of catalysis (2-4).Various experimental techniques allow monitoring changes in interactions resulting from configurational changes. Long-range micro/millisecond domain motions in flexible proteins have been studied by NMR (5) and FRET techniques (6, 7). Molecula...

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“…Ser88 has been proposed to act as a nucleophile in the catalytic reaction although not properly positioned for a nucleophilic attack in the crystal structure [5,6]. Preventing the binding of dUMP to the enzyme active site significantly inhibits the NAD(P)H-oxidase activity [9] and the dUMP binding site is recognized as a target for ThyX inhibitors. ThyX subunits adopt an identical hammerhead sharkshaped overall structure in PBCV-1 and TmThyX [7] consisting of a central domain of curved four-stranded anti-parallel ␤-sheets and six helices; small variations in the orientation of the helices that are not in direct contact with the ␤-sheet of the central core domain between the TmThyX and PBCV-1 proteins exist [7].…”

Section: Introductionmentioning

confidence: 99%