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

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 substit...