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.

中文翻译：

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醛脱氢酶1A（ALDH1A）亚家族成员的结构和动力学特征，在维甲酸生物合成中具有活性的癌症干细胞标志物。

醛脱氢酶催化NAD（P）+依赖性的醛氧化为相应的羧酸。最近获得了人ALDH1A酶的三维结构，但缺乏在相同实验条件下的完整动力学特征。我们显示三种酶，ALDH1A1，ALDH1A2和ALDH1A3，具有相似的拓扑结构，尽管在其底物结合口袋中的体积有所减少。使用改进的基于HPLC的视黄醛方法并列表征了脂族和类视黄醛醛的活性。己醛是最有效的底物。与ALDH1A2和ALDH1A3相比，ALDH1A1在己醛，反-2-己醛和柠檬醛中显示出较低的Km值。以kcat / Km计，ALDH1A2是脂质过氧化产物4-羟基-2-壬烯醛的最佳酶。对全反式和9-顺式-视黄醛的催化效率通常低于链烷烃和链烯醛。ALDH1A2和ALDH1A3对全反式维甲酸具有较高的催化效率。ALDH1A3对9-顺式-视黄醛对全反式异构体的特异性较低可能与其底物结合口袋的体积较小有关。镁抑制ALDH1A1和ALDH1A2，而镁激活ALDH1A3，这与辅酶解离是ALDH1A1和ALDH1A2的限速步骤，以及ALDH1A3脱酰作用，以己醛为底物。我们突变了ALDH1A1（L114P）和ALDH1A2（N475G，A476V，L477V，N478S），以模拟它们对应的底物结合口袋。ALDH1A1对柠檬醛的特异性可追溯至残基114和残基458至461。关于视黄醛，