Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Barcelona, Spain.
Chem Biol Interact. 2011 May 30;191(1-3):199-205. doi: 10.1016/j.cbi.2011.02.007. Epub 2011 Feb 15.
NADP(H)-dependent cytosolic aldo-keto reductases (AKR) are mostly monomeric enzymes which fold into a typical (α/β)(8)-barrel structure. Substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable loops (A, B, and C). Based on sequence identity, AKR have been grouped into families, namely AKR1-AKR15, containing multiple subfamilies. Two human enzymes from the AKR1B subfamily (AKR1B1 and AKR1B10) are of special interest. AKR1B1 (aldose reductase) is related to secondary diabetic complications, while AKR1B10 is induced in cancer cells and is highly active with all-trans-retinaldehyde. Residues interacting with all-trans-retinaldehyde and differing between AKR1B1 and AKR1B10 are Leu125Lys and Val131Ala (loop A), Leu301Val, Ser303Gln, and Cys304Ser (loop C). Recently, we demonstrated the importance of Lys125 as a determinant of AKR1B10 specificity for retinoids. Residues 301 and 304 are also involved in interactions with substrates or inhibitors, and thus we checked their contribution to retinoid specificity. We also extended our study with retinoids to rodent members of the AKR1B subfamily: AKR1B3 (aldose reductase), AKR1B7 (mouse vas deferens protein), AKR1B8 (fibroblast-growth factor 1-regulated protein), and AKR1B9 (Chinese hamster ovary reductase), which were tested against all-trans isomers of retinaldehyde and retinol. All enzymes were active with retinaldehyde, but with k(cat) values (0.02-0.52 min(-1)) much lower than that of AKR1B10 (27 min(-1)). None of the enzymes showed oxidizing activity with retinol. Since these enzymes (except AKR1B3) have Lys125, other residues should account for retinaldehyde specificity. Here, by using site-directed mutagenesis and molecular modeling, we further delineate the contribution of residues 301 and 304. We demonstrate that besides Lys125, Ser304 is a major structural determinant for all-trans-retinaldehyde specificity of AKR1B10.
NADP(H)-依赖的细胞质醛酮还原酶 (AKR) 大多是单体酶，它们折叠成典型的 (α/β)(8)-桶状结构。底物特异性和抑制剂选择性由与位于三个高度可变环 (A、B 和 C) 中的残基相互作用决定。根据序列同一性，AKR 已被分为家族，即 AKR1-AKR15，包含多个亚家族。AKR1B 亚家族的两种人类酶 (AKR1B1 和 AKR1B10) 特别引人注目。AKR1B1(醛糖还原酶)与二级糖尿病并发症有关，而 AKR1B10 在癌细胞中诱导，对全反式视黄醛具有高度活性。与 AKR1B1 和 AKR1B10 相互作用并不同的残基是 Leu125Lys 和 Val131Ala(环 A)、Leu301Val、Ser303Gln 和 Cys304Ser(环 C)。最近，我们证明了 Lys125 作为 AKR1B10 对类视黄醇特异性的决定因素的重要性。残基 301 和 304 也参与与底物或抑制剂的相互作用，因此我们检查了它们对类视黄醇特异性的贡献。我们还将我们的研究扩展到 AKR1B 亚家族的啮齿动物成员：AKR1B3(醛糖还原酶)、AKR1B7(小鼠输精管蛋白)、AKR1B8(成纤维细胞生长因子 1 调节蛋白)和 AKR1B9(中国仓鼠卵巢还原酶)，它们都针对全反式视黄醛和视黄醇的异构体进行了测试。所有酶都对视黄醛有活性，但 k(cat) 值(0.02-0.52 min(-1))远低于 AKR1B10(27 min(-1))。没有一种酶对视黄醇表现出氧化活性。由于这些酶(AKR1B3 除外)都有 Lys125，其他残基应该是视黄醛特异性的原因。在这里，我们通过定点突变和分子建模，进一步描绘了残基 301 和 304 的贡献。我们证明，除了 Lys125 之外，Ser304 也是 AKR1B10 全反式视黄醛特异性的主要结构决定因素。
