Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
Arch Biochem Biophys. 2012 Nov 1;527(1):23-30. doi: 10.1016/j.abb.2012.07.012. Epub 2012 Aug 1.
In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo-keto reductase (AKR)(1) 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/β-dihydro-C19/C21/C24-steroids into the corresponding 3β-hydroxysteroids, showing K(m) of 1.3-9.1 μM and k(cat) of 1.1-7.6 min(-1). The stereospecific reduction was also observed in the metabolism of 5α- and 5β-dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3β-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate.
在这项研究中，我们分离了一种兔醛糖还原酶样蛋白的 cDNA，它与人类醛酮还原酶（aldo-keto reductase）（1）1B10 有 86%的序列同一性，并在 AKR 超家族中被指定为 AKR1B19。纯化的重组 AKR1B19 在对各种醛和α-二羰基化合物的底物特异性方面与 AKR1B10 和兔醛糖还原酶（AKR1B2）相似。与 AKR1B10 和 AKR1B2 不同，AKR1B19 有效地将 3-酮-5α/β-二氢-C19/C21/C24-甾体还原为相应的 3β-羟基甾体，显示 K(m)为 1.3-9.1 μM 和 k(cat)为 1.1-7.6 min(-1)。在 AKR1B19 过表达细胞中，5α-和 5β-二氢睾丸酮的代谢也观察到立体特异性还原。AKR1B19 的 mRNA 在兔组织中广泛表达，并且该酶与肺中的 3β-羟甾酮脱氢酶活性一起被共纯化。因此，AKR1B19 可能作为 3-酮还原酶，以及兔组织中细胞毒性羰基化合物的防御系统发挥作用。通过分别用 Gln 和 Ser 替换 AKR1B10 中的 Phe303 和 Met304，研究了 AKR1B19 独特的 3-酮还原酶活性的分子决定因素。单突变（F303Q、M304S）和双突变（F303Q/M304S）显著损害了这种活性，表明这两个残基在识别甾体底物中起关键作用。
