Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.
|Journal||Archives of biochemistry and biophysics|
|Publication status||Published - 15 Mar 2020|
- Aldehyde dehydrogenase
- Retinoic acid
- Substrate-binding pocket