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.
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Submitted on : Wednesday, February 5, 2014 - 4:13:33 PM
Last modification on : Thursday, February 7, 2019 - 2:29:25 PM

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Sergey Laptenok, Latifa Bouzhir-Sima, Jean-Christophe Lambry, Hannu Myllykallio, Ursula Liebl, et al.. Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX.. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2013, 110 (22), pp.8924-8929. ⟨10.1073/pnas.1218729110⟩. ⟨hal-00942352⟩

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