Enzyme Active Site Loop Revealed as a Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-Based Kinetics - Université de Lorraine Accéder directement au contenu
Article Dans Une Revue ACS Catalysis Année : 2019

Enzyme Active Site Loop Revealed as a Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-Based Kinetics

Résumé

Structural motions are key events in enzyme catalysis, as exemplified by the conformational dynamics associated with the cofactor in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases. We previously showed that, after the oxidoreduction step, the reduced cofactor must adopt a flipped conformation, which positions the nicotinamide in a conserved cavity that might constitute the exit door for NAD(P)H. However, the molecular basis that make this movement possible is unknown. Based on the pre- and postflip X-ray structures, targeted molecular dynamic simulations enabled us to identify the E268LGG271 conserved loop that must shift to allow reduced nicotinamide conformational switch. To monitor cofactor movements within the active site, we used an intrinsic fluorescence resonance energy transfer signal between Trp177 and the reduced nicotinamide moiety to kinetically track the flip during the catalytic cycle of retinal dehydrogenase 2 (ALDH1A2). Decreasing loop flexibility by substituting Ala for Gly271 drastically reduced the rate constant associated with this movement that became rate-limiting. We thus propose that the E268LGG271 loop acts as a gatekeeper for cofactor flipping. Similar approaches applied to a CoA-dependent aldehyde dehydrogenase showed that cofactor flipping likely extends to the whole ALDH family, thus bridging the gap between the well-studied chemical steps and a conformational transition essential for catalysis.
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Dates et versions

hal-02022828 , version 1 (23-11-2020)

Identifiants

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Sophie Rahuel-Clermont, Raphaël Bchini, Sophie Barbe, Séverine Boutserin, Isabelle André, et al.. Enzyme Active Site Loop Revealed as a Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-Based Kinetics. ACS Catalysis, 2019, 9 (2), pp.1337-1346. ⟨10.1021/acscatal.8b03951⟩. ⟨hal-02022828⟩
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