Crystal structure and catalytic characterization of the dehydrogenase/reductase SDR family member 4 (DHRS4) from Caenorhabditis elegans
Crystal structure and catalytic characterization of the dehydrogenase/reductase SDR family member 4 (DHRS4) from Caenorhabditis elegans
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DOI：
10.1111/febs.14337
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发表时间：
2018-01-01
期刊：
影响因子：
5.4
通讯作者：
Scheidig, Axel J.
中科院分区：
文献类型：
--
作者：
Kisiela, Michael;Faust, Annette;Scheidig, Axel J.
人脱氢酶/还原酶SDR家族成员4（DHRS 4）是参与几种芳香族羰基化合物、类固醇和胆汁酸的代谢的四聚体蛋白。迄今为止唯一被表征的无脊椎动物DHRS 4是来自模式生物秀丽隐杆线虫的DHRS 4。我们先前已经克隆并初步表征了这种蛋白质，其最近在UniProtKB数据库中被注释为DHRS4_CAEEL。与双乙酰复合的全长DHRS4_CAEEL蛋白的结晶和X-射线衍射研究揭示了其四聚体结构，并表明两个亚基通过分子间二硫键连接，该二硫键由每条蛋白链的N-末端半胱氨酸残基（Cys5）形成，这增加了酶活性。更详细的生物化学和催化表征表明，DHRS4_CAEEL与人DHRS4具有一些相同的特性，例如与脂肪族α-二酮的底物亲和力相对较低，以及对芳香族二羰基化合物（如靛红）的偏好，与人酶相比Km值低30倍。此外，DHRS4_CAEEL对脂肪族醛（例如己醛）具有活性，而人DHRS4则没有。脱氢酶活性与醇只观察到与芳香醇。蛋白质热位移分析显示磷酸盐缓冲液的稳定作用，伴随着催化活性的增加超过两倍。DHRS 4同源基因在简单谱系如C.秀丽线虫的研究可能有助于我们理解这种蛋白质的原始功能，这种蛋白质是在从无脊椎动物发展到高等哺乳动物物种的过程中通过进化过程形成的。
The human dehydrogenase/reductase SDR family member 4 (DHRS4) is a tetrameric protein that is involved in the metabolism of several aromatic carbonyl compounds, steroids, and bile acids. The only invertebrate DHRS4 that has been characterized to date is that from the model organism Caenorhabditis elegans. We have previously cloned and initially characterized this protein that was recently annotated as DHRS4_CAEEL in the UniProtKB database. Crystallization and X-ray diffraction studies of the full-length DHRS4_CAEEL protein in complex with diacetyl revealed its tetrameric structure and showed that two subunits are connected via an intermolecular disulfide bridge that is formed by N-terminal cysteine residues (Cys5) of each protein chain, which increases the enzymatic activity. A more detailed biochemical and catalytic characterization shows that DHRS4_CAEEL shares some properties with human DHRS4 such as relatively low substrate affinities with aliphatic alpha-diketones and a preference for aromatic dicarbonyls such as isatin, with a 30-fold lower Km value compared with the human enzyme. Moreover, DHRS4_CAEEL is active with aliphatic aldehydes (e.g. hexanal), while human DHRS4 is not. Dehydrogenase activity with alcohols was only observed with aromatic alcohols. Protein thermal shift assay revealed a stabilizing effect of phosphate buffer that was accompanied by an increase in catalytic activity of more than two-fold. The study of DHRS4 homologs in simple lineages such as C. elegans may contribute to our understanding of the original function of this protein that has been shaped by evolutionary processes in the course of the development from invertebrates to higher mammalian species.