Human NRDRB1, an Alternatively Spliced Isoform of NADP(H)-Dependent Retinol Dehydrogenase/Reductase Enhanced Enzymatic Activity of Benzil
Human NRDRB1, an Alternatively Spliced Isoform of NADP(H)-Dependent Retinol Dehydrogenase/Reductase Enhanced Enzymatic Activity of Benzil
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DOI：
10.1159/000343326
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发表时间：
2012-11
影响因子：
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通讯作者：
Yinxia Yan;Xuhong Song;Gefei Liu;Z. Su;Yongming Du;Xuxia Sui;X. Chang;Dongyang Huang
中科院分区：
文献类型：
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作者：
Yinxia Yan;Xuhong Song;Gefei Liu;Z. Su;Yongming Du;Xuxia Sui;X. Chang;Dongyang Huang
目的：人NRDRB 1是NADP（H）依赖性视黄醇脱氢酶/还原酶（NRDR）的一种226个氨基酸的选择性剪接异构体，缺乏外显子3的完整编码区，但保留了NRDR催化活性的所有重要功能基序。然而，其组织分布和生理功能仍有待阐明。方法：采用半定量PCR和western blot方法分析NRDRB 1和NRDR在细胞和组织中的表达。NRDRB 1表达为His 6融合蛋白，并进行动力学测定。结果：重组NRDRB 1的kcat/Km值比重组NRDR高1.2至8.6倍，取决于底物。NRDRB 1催化α-二羰基化合物的NADPH依赖性还原，如靛红，9，10-菲醌，特别是苯偶酰。NRDRB 1在人肝脏中的显著高催化活性和相对高的表达赋予细胞对苯偶酰诱导的细胞毒性的抗性，并且在低表达的Ec 109细胞中过表达NRDRB 1显著增强细胞对苯偶酰的耐受性。结论：基于其底物特异性、催化活性和在人肝组织中相对高的表达，我们的研究结果表明，NRDRB 1，一种NRDR在体内的选择性剪接异构体，作为含有活性羰基的异生物质的二羰基还原酶，其功能优于NRDR。我们的研究是第一次报道这种现象的酶参与生化反应。
Aims: Human NRDRB1, a 226 amino acid alternatively spliced isoform of the NADP(H)- dependent retinol dehydrogenase/reductase (NRDR), lacks the complete coding region of exon 3, but preserves all the important functional motifs for NRDR catalytic activity. Nevertheless, its tissue distribution and physiological function remain to be elucidated. Methods: Expression of NRDRB1 and NRDR in cells and tissues was analyzed by semi-quantitative polymerase chain reaction (PCR) and western blot. NRDRB1 was expressed as a His6 fusion protein and subjected to kinetics assays. Results: Recombinant NRDRB1 had 1.2 to 8.6 fold higher kcat/Km values than recombinant NRDR, depending on the substrate. NRDRB1 catalyzed the NADPHdependent reduction of α-dicarbonyl compounds, such as isatin, 9,10-phenanthrenequinone, and especially benzil. The significantly high catalytic activity and the relatively high expression in human liver of NRDRB1 conferred cellular resistance to benzil-induced cell toxicity and over-expression of NRDRB1 in low expressing Ec109 cells significantly enhanced cell tolerance toward benzil. Conclusions: Based on its substrate specificity, catalytic activity and relatively high expression in human liver tissue, our results suggest that NRDRB1, an alternatively spliced isoform of NRDR in vivo functions better than NRDR as a dicarbonyl reductase for xenobiotics containing reactive carbonyls. Our study is the first reporting this phenomenon of the enzymes involved in biochemical reactions.