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

Abstract

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.

Keywords: flavoprotein; protein dynamics; quenching; ultrafast fluorescence spectroscopy.

Fig. 1.

(A) Transient fluorescence spectra of WT TmThyX measured at 20 °C. The feature at ∼460 nm in the 0-ps spectrum is due to Raman scattering of water. (B) Kinetics at 520 nm of WT and Y91F ThyX. The time axis is linear until 10 ps and logarithmic thereafter. Solid lines are fits with a four-parameter exponential decay; the dashed line with power law.

Fig. 2.

Temperature dependence of peak-normalized fluorescence decay of WT TmThyX at 520 nm. The time axis is linear until 10 ps and logarithmic thereafter.

Fig. 3.

Analysis of the temperature dependence of the fluorescence kinetics of WT TmThyX using Eq. S1. (A) Mean value τ₀ of the lifetime distribution. (B) Heterogeneity factor q of the lifetime distribution. The solid lines are guides for the eye. (C) Lifetimes distribution. (D) Distribution of edge-to-edge distances between FAD and electron donors normalized to the total integral.

Fig. 4.

Effect of dUMP binding on fluorescence decay of WT and Y91F TmThyX at 520 nm. The time axis is linear until 4 ps and logarithmic thereafter. A global analysis in terms of DAS of WT TmThyX fluorescence spectra in the presence of dUMP is shown in Fig. S3F.

Fig. 5.

Superimposed structures of elements of the four active sites, time averaged over the last 500 ps of the free trajectories of MD simulations of ThyX in the presence (A) and absence (B) of dUMP. The superposition is on the entire backbone of each subunit. The His53 residues associated with the active sites belong to different subunits as the other elements. The carbon atoms are color-coded with the respective subunits.

Fig. 6.

Dynamics (Left) and distribution histograms (Right) of the distance between FAD and Tyr-91 (shortest distance between an atom on the isoalloxazine ring and the tyrosine aromatic ring) in the four subunits during the 2.5-ns free dynamics.