Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX Sergey P. Laptenoka,b, Latifa Bouzhir-Simaa,b, Jean-Christophe Lambrya,b, Hannu Myllykallioa,b, Ursula Liebla,b, and Marten H. Vosa,b,1 aLaboratory for Optics and Biosciences, Centre National de la Recherche Scientifique Ecole Polytechnique, 91128 Palaiseau, France; and bInstitut National de la Santé et de la Recherche Médicale U696, 91128 Palaiseau, France Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved April 18, 2013 (received for review November 9, 2012) In many bacteria the flavoenzyme thymidylate synthase ThyX optical techniques. In particular, the fluorescence properties of produces the DNA nucleotide deoxythymidine monophosphate intrinsic or external fluorophores can be exquisitely sensitive to from dUMP, using methylenetetrahydrofolate as carbon donor the protein environment. In flavoproteins, interaction of the and NADPH as hydride donor. Because all three substrates bind in flavin cofactor with the protein environment has been shown to close proximity to the catalytic flavin adenine dinucleotide group, diminish the lifetime of the flavin fluorescence from the intrinsic substantial flexibility of the ThyX active site has been hypothe- nanosecond timescale to the femtoseconds-to-picoseconds time- sized. Using femtosecond time-resolved fluorescence spectros- scale, due to quenching by photooxidation of nearby aromatic copy, we have studied the conformational heterogeneity and the residues (3, 9–15). In the present study, we use ultrafast fluo- conformational interconversion dynamics in real time in ThyX from rescence spectroscopy to investigate conformational flexibility of the hyperthermophilic bacterium Thermotoga maritima. The dy- the flavoenzyme thymidylate synthase ThyX. namics of electron transfer to excited flavin adenine dinucleotide ThyX is a homotetrameric enzyme discovered a decade ago (16), from a neighboring tyrosine residue are used as a sensitive probe which is essential for de novo synthesis of the DNA precursor 2′- of the functional dynamics of the active site. The fluorescence de- deoxythymidine-5′-monophosphate (dTMP) in a large number of cay spanned a full three orders of magnitude, demonstrating a very bacterial systems. ThyX shows no structural homology to thymi- wide range of conformations. In particular, at physiological temper- dylate synthase ThyA, which is used in most eukaryotes (17). Be- BIOPHYSICS AND atures, multiple angstrom cofactor-residue displacements occur on cause the ThyX pathway is used by a number of pathogenic bacteria fi COMPUTATIONAL BIOLOGY the picoseconds timescale. These experimental ndings are sup- and absent in humans, ThyX is considered a promising antimicrobial ported by molecular dynamics simulations. Binding of the dUMP target (16, 18); it catalyses carbon transfer from N5,N10-methylene- fl substrate abolishes this exibility and stabilizes the active site in 5,6,7,8-tetrahydrofolate (MTHF or CH H folate) to deoxyuridine fi fl 2 4 acon guration where dUMP closely interacts with the avin co- monophosphate (dUMP) using NADPH as a hydride donor and factor and very efficiently quenches fluorescence itself. Our results fi consequently has three substrates (dUMP, MTHF, NADPH) with indicate a dynamic selected- t mechanism where binding of the the flavin adenine dinucleotide cofactor shuttling between the first substrate dUMP at high temperature stabilizes the enzyme fully oxidized (FAD) and fully reduced (FADH ) forms. dUMP in a configuration favorable for interaction with the second sub- 2 binds in close interaction with the flavin group, displacing a nearby strate NADPH, and more generally have important implications for Tyr residue (19). A very recent study shows that folate derivatives the role of active site flexibility in enzymes interacting with multiple may bind to the opposite side of the flavin cofactor with respect to poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active dU(20). The binding site of NADPH has not been determined by site dynamics of ThyX and other multisubstrate flavoenzymes. structural studies, but inhibition studies indicate that folate and NADPH binding sites may partially coincide (21). Furthermore, fl protein dynamics | flavoprotein | ultrafast fluorescence spectroscopy | reduction of avin by NADPH appears gated by the presence of quenching dUMP (21), further pointing at substrate-induced, functional structural rearrangements. Steady-state crystallographic studies of fl fi fl this enzyme also indicate substantial exibility of the active site: in on gurational exibility is essential for enzyme function the substrate-free structure the flavin group and its close environ- Cduring catalysis. Binding of one or more substrates, accom- ment appear disordered (19); for the folate-bound form, multiple modation of the transition state where the actual reaction takes flavin configurations have been suggested (20). place, relaxation to the product state, and release of the product fl fi Using a newly developed, ultrafast uorescence spectrometer (s) is possible because different con gurations of the enzyme are with full spectral resolution, we performed studies on the dynamics continuously sampled, by thermal or reaction-driven motions, of FAD fluorescence in wild-type and genetically modified ThyX on timescales ranging from femtoseconds to microseconds. The fi enzymes from several bacterial species. In the present work we con gurational space sampled in a certain time range will de- focus on the enzyme from the hyperthermophilic bacterium Ther- pend on the local protein flexibility/energy landscape and the fi motoga maritima that allows studies over a wide temperature range. temperature (1). During these con gurational changes, distances We identified a close-lying tyrosine as well as the substrate dUMP between constituents of the enzyme complex change. It has been itself as fluorescence quenchers. The observed fluorescence decay recognized that the fastest (femtosecond to picosecond) local- revealed the presence of a wide range of conformations, whose ized motions exist alongside the slower motions that occur on the (typically millisecond) timescale of catalysis (2–4). Various experimental techniques allow monitoring changes in Author contributions: H.M., U.E.L., and M.H.V. designed research; S.P.L., L.B.-S., and J.-C.L. interactions resulting from configurational changes. Long-range performed research; S.P.L. contributed new reagents/analytic tools; S.P.L., J.-C.L., and micro/millisecond domain motions in flexible proteins have been M.H.V. analyzed data; and S.P.L., H.M., U.E.L., and M.H.V. wrote the paper. studied by NMR (5) and FRET techniques (6, 7). Molecular dy- The authors declare no conflict of interest. namics simulations and NMR experiments indicate that more lo- This article is a PNAS Direct Submission. calized structural fluctuations in these flexible regions occur on the 1To whom correspondence should be addressed. E-mail: [email protected]. fl picoseconds-to-nanoseconds timescale (6, 8). These uctuations This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can only be directly investigated using very high time-resolution 1073/pnas.1218729110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218729110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 interconversion accelerates at the physiological temperature of this designed to prevent electron transfer while maximally preserving hyperthermophilic enzyme. Binding of dUMP was found to stabi- the structural environment of the flavin. lize the active site in a configuration allowing close interaction The overall fluorescence decay kinetics of the mutant ThyX between dUMP and FAD and favorable for interaction with protein are much slower than those of WT (Fig. 1B; note the NADPH. Our data have important implications for the role of active logarithmic scaling after 10 ps), implying that ET from Tyr-91 is site flexibility in multisubstrate enzymes and ultimately permit ex- indeed the dominant quenching process. However, in both cases, ploring the effect of inhibitor molecules on the active site dynamics. a fast decay in the order of ∼1 ps is present (see below), which we assign to relaxation processes in the excited state. Furthermore, Results the decay of the mutant protein still takes place faster than that The absorption spectrum of ThyX from T. maritima (TmThyX; of FAD in solution, indicating that aromatic residues other than fl Fig. S1A) is very similar to that published previously (22), and the Tyr-91 also contribute to quenching. The shape of the uores- ∼ fluorescence spectrum (Fig. S1C) typical for oxidized flavins. The cence spectra was found to be constant after 200 ps and to be significantly different from that of FAD in solution (Fig. S3A). enzyme contains four FAD binding sites (closest center-to-center fi distance 27 Å). Because in principle, resonance energy transfer This observation further con rms that free FAD does not con- between identical molecules within the protein (homo-FRET) can tribute to the signal. Analysis of the full data set in terms of multiexponential decay of take place, potentially complicating the analysis, we measured both the WT and the Y91F mutant protein requires at least four fluorescence anisotropy. The anisotropy was close to the theo- decay rates (Fig. S3 A and B). This analysis indicates that red shifts retical maximum of 0.4,