Characterization of rat and mouse NAD+-dependent 3α/17β/20α-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis
Characterization of rat and mouse NAD+-dependent 3α/17β/20α-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis
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DOI：
10.1016/j.abb.2007.08.011
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发表时间：
2007-11-01
影响因子：
3.9
通讯作者：
Hara, Akira
中科院分区：
文献类型：
--
作者：
Endo, Satoshi;Sanai, Masaharu;Hara, Akira
在这项研究中，我们鉴定了大鼠和小鼠的醛酮还原酶（分别为AKR1C16和AKR1C13）具有92%的序列一致性。重组酶以NAD（+）为首选辅酶氧化非甾体醇，表现出较低的3 α /17 β /20 α -羟基类固醇脱氢酶（HSD）活性。底物特异性与大鼠NAD（+）依赖性3 α - hsd （AKR1C17）不同，后者与AKR1C16具有95%的序列一致性。为了阐明决定酶底物特异性的残基，我们对AKR1C16的Tyr24、Asp128和Phe129与AKR1C17的相应残基（分别为Ser、Tyr和Leu）进行了定点突变。双突变（Asp128/ tyrr - phe129 /Leu）对底物特异性影响不大，而Tyr24/Ser突变体仅显示3 α - hsd活性，三个残基的三重突变产生的酶几乎与AKR1C17具有相同的性质。AKR1C17的Ser24/Tyr突变导致3 α - hsd活性降低，17 β -和20 α - hsd活性出现，证实了残基24对底物识别的重要性。AKR1C16与大鼠NAD（+）依赖性17 β - hsd （AKR1C24）也有92%的相同，后者具有Tyr24。用AKR1C24的相应残基（分别为Glu、Ser和Phe）取代AKR1C16的Asp128、Phel29和Ser137，提高了17个β -和20个α -羟基类固醇的催化效率。(c) 2007爱思唯尔公司版权所有。
In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD(+) as the preferred coenzyme, and showed low 3 alpha/17 beta/20 alpha-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD(+)-dependent 3 alpha-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3 alpha-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3 alpha-HSD activity and appearance of 17 beta- and 20 alpha-HSD activities. AKR1C16 is also 92% identical with rat NAD(+)-dependent 17 beta-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phel29 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17 beta- and 20 alpha-hydroxysteroids. (c) 2007 Elsevier Inc. All rights reserved.