Characterization of a rabbit morphine 6-dehydrogenase and its two isoforms which function as NAD+-dependent 3α(17β)- hydroxysteroid dehydrogenases
Characterization of a rabbit morphine 6-dehydrogenase and its two isoforms which function as NAD+-dependent 3α(17β)- hydroxysteroid dehydrogenases
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兔吗啡 6-脱氢酶及其作为 NAD+ 依赖性 3α(17β)-羟基类固醇脱氢酶发挥作用的两种亚型的表征
DOI：
10.1016/j.abb.2012.11.013
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Akira Hara
中科院分区：
文献类型：
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作者：
Satoshi Endo;Toshiyuki Matsunaga;Airi Fujimoto;Sho Kumada;Yuki Arai;Yoko Miura;Hiroshige Mikamo;Ossama El-Kabbani;Shigeru Yamano;Munekazu Iinuma;Akira Hara
哺乳动物吗啡6-脱氢酶（M6DH）1将吗啡转化为反应性亲电试剂吗啡酮。M6DH属于醛酮还原酶（AKR）超家族，但其内源性底物和整个氨基酸序列尚不清楚。最近的一项兔基因组测序预测，新的AKRs的三个基因（1C26, 1C27和1C28）具有bbbb87 %的氨基酸序列一致性，并且与兔肝脏M6DH的部分序列相似。我们分离了这三种akr的cdna，并比较了它们的重组酶的性质。与M6DH相似，只有AKR1C26与肝脏M6DH具有最高序列同一性。这3种AKRs对其他非甾体脂环醇和3α/17β-羟基- c18 /C19/ c21类固醇具有NAD+依赖性脱氢酶活性，其mrna在家兔组织中普遍表达。底物的动力学常数表明，AKR1C26和AKR1C28至少是NAD+依赖性的3α/17β-羟基类固醇脱氢酶。AKR1C27与AKR1C28的不同之处在于其对底物的高km值和对竞争性抑制剂（鸢尾苷A、松木酚、己甾醇和玉米赤霉烯酮）的低敏感性，尽管它们的序列具有95%的一致性。将AKR1C27中的Tyr118和Phe310位点突变为AKR1C28中相应的残基（分别为Phe和Ile），产生了一种与AKR1C28相似的酶，表明它们在配体结合中起关键作用。
Mammalian morphine 6-dehydrogenase (M6DH)1converts morphine into a reactive electrophile, morphinone. M6DH belongs to the aldo–keto reductase (AKR) superfamily, but its endogenous substrates and entire amino acid sequence remain unknown. A recent rabbit genomic sequencing predicts three genes for novel AKRs (1C26, 1C27 and 1C28) that share >87% amino acid sequence identity and are similar to the partial sequence of rabbit liver M6DH. We isolated cDNAs for the three AKRs, and compared the properties of their recombinant enzymes. Like M6DH, only AKR1C26 that shares the highest sequence identity with hepatic M6DH oxidized morphine. The three AKRs showed NAD+-dependent dehydrogenase activity towards other non-steroidal alicyclic alcohols and 3α/17β-hydroxy-C18/C19/C21-steroids, and their mRNAs were ubiquitously expressed in rabbit tissues. The kinetic constants for the substrates suggest that at least AKR1C26 and AKR1C28 act as NAD+-dependent 3α/17β-hydroxysteroid dehydrogenases. AKR1C27 differed from AKR1C28 in its high Kmvalues for the substrates and low sensitivity towards competitive inhibitors (ikarisoside A, hinokitiol, hexestrol and zearalenone), despite their 95% sequence identity. The site-directed mutagenesis of Tyr118 and Phe310 in AKR1C27 to the corresponding residues (Phe and Ile, respectively) in AKR1C28 produced an enzyme that was similar to AKR1C28, suggesting their key roles in ligand binding.