Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases

Abstract

. The submicromolar Km of ALDH1 for all-trans retinal, and its 600-fold enhanced affinity for retinal compared to acetaldehyde, are explained by the size and shape of the substrate entrance tunnel in ALDH1. All-trans retinal fits into the active-site pocket of ALDH1, but not into the pocket of ALDH2. Two helices and one surface loop that line the tunnel are likely to have a key role in defining substrate specificity in the wider ALDH family. The relative sizes of the tunnels also suggest why the bulky alcohol aversive drug disulfiram reacts more rapidly with ALDH1 than ALDH2. The disorder of Glu268 and the observation that NAD+ binds in two distinct modes indicate that flexibility is a key facet of the enzyme reaction mechanism.

Conserved Catalytic Residues of the ALDH1L1 Aldehyde Dehydrogenase Domain Control Binding and Discharging of the Coenzyme

“…After the hydride transfer, the reduced nicotinamide ring must exit the catalytic site to allow for the accessibility of a water molecule, which hydrolyzes the acyl-sulfur bond releasing the product. In agreement with this mechanism, numerous crystal structures of ALDH revealed two common conformations of the coenzyme, depending on its oxidation state: the extended ("hydride transfer") conformation of NAD(P) ϩ , with the nicotinamide ring positioned close to Cys-302; and the contracted ("hydrolysis") conformation of NAD(P)H, with the nicotinamide ring found outside of the catalytic site or disordered (4,5,7,10,25,27,30). The mechanism by which ALDHs sense the oxidation state of the bound coenzyme and control its conformation is not fully understood.…”

“…Catalytic Glutamate Is Critical for Mobility and Correct Positioning of Coenzyme-Several studies have suggested that the invariant catalytic glutamate of ALDHs (Glu-268 in class 1 and 2 enzymes) participates in both acylation and deacylation stages of the ALDH catalysis (5,7,23,25). Specifically, it has been proposed that in the first step it deprotonates the catalytic cysteine prior to nucleophilic attack on the substrate, whereas in the second step it activates a water molecule to facilitate the hydrolysis of the acyl-enzyme intermediate.…”

“…Active Site Cysteine Distinguishes between Oxidized and Reduced Forms of Coenzyme-Although numerous studies have demonstrated that oxidized and reduced forms of the coenzyme are bound to ALDHs in distinct conformations (5,7,10,(27)(28)(29), the mechanism of recognition of NAD(P) ϩ versus NAD(P)H remains unclear. In our study, in the structures of the binary complexes of the C707A mutant of C t -FDH with NADP ϩ or NADPH, the coenzymes were bound in the same (extended) conformation with the nicotinamide moieties clearly present in the active site, which is typically observed only for NAD(P) ϩ (Fig.…”

“…In the second step, an activated water molecule hydrolyzes the thioester, releasing the product. It has been suggested that deprotonation of the catalytic cysteine prior to the nucleophilic attack on the substrate, a required step in this mechanism, involves acceptance of a proton by either glutamate 268 or 399 (5,7,20,(22)(23)(24). The resulting thiolate ion is likely stabilized by the positively charged nic-otinamide ring of the coenzyme and/or adjacent main chain amide groups.…”

“…It has also been suggested that, in tetrameric class 1 and 2 ALDHs, the proton abstracted by Glu-268 is shuttled to bulk water (10,25). In addition, Glu-268 is also the most likely residue activating a water molecule in the deacylation step of the reaction (5,7,10,23,26).…”