Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic ă effects throughout life in vertebrate organisms. Thus, RA action must be ă tightly regulated through the coordinated action of biosynthetic and ă degrading enzymes. The last step of retinoic acid biosynthesis is ă irreversibly catalyzed by the NAD-dependent retinal dehydrogenases ă (RALDH), which are members of the aldehyde dehydrogenase (ALDH) ă superfamily. Low intracellular retinal concentrations imply efficient ă substrate molecular recognition to ensure high affinity and specificity ă of RALDHs for retinal. This study addresses the molecular basis of ă retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or ă RALDH2), through the comparison of the catalytic behavior of retinal ă analogs and use of the fluorescence properties of retinol. We show that, ă in contrast to long chain unsaturated substrates, the rate-limiting step ă of retinal oxidation by RALDHs is associated with acylation. Use of the ă fluorescence resonance energy transfer upon retinol interaction with ă RALDHs provides evidence that retinal recognition occurs in two steps: ă binding into the substrate access channel, and a slower structural ă reorganization with a rate constant of the same magnitude as the k(cat) ă for retinal oxidation: 0.18 vs. 0.07 and 0.25 vs. 0.1 s(-1) for ALDH1A1 ă and ALDH1A2, respectively. This suggests that the conformational ă transition of the RALDH-retinal complex significantly contributes to the ă rate-limiting step that controls the kinetics of retinal oxidation, as a ă prerequisite for the formation of a catalytically competent Michaelis ă complex. This conclusion is consistent with the general notion that ă structural flexibility within the active site of ALDH enzymes has been ă shown to be an integral component of catalysis. (C) 2012 Elsevier ă Ireland Ltd. All rights reserved.