Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School, Schleswig-Holstein, Campus Kiel, Brunswiker Strasse 10, D-24105 Kiel, Germany.
J Steroid Biochem Mol Biol. 2012 Nov;132(3-5):203-11. doi: 10.1016/j.jsbmb.2012.05.012. Epub 2012 Jun 5.
"3α-Hydroxysteroid dehydrogenase/carbonyl reductase regulator" (HsdR) from Comamonas testosteroni (C. testosteroni) was identified as a member of the LysR-type transcriptional regulator (LTTR) family. We have shown previously that HsdR activates the expression of the hsdA gene, encoding 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR), which is an important enzyme involved in the degradation of steroid compounds. Phylogenetic analysis indicated that HsdR is related to the contact-regulated gene A (CrgA) from Neisseria meningitidis, which exists as a homooctamer. Therefore, to further elucidate the regulatory mechanism of HsdR, we investigated the oligomeric state and autoregulation of this transcriptional factor in the present study. To identify the active domains of HsdR, three truncated forms, HsdRΔN (N-terminus deleted), HsdRΔC (C-terminus deleted), and HsdRΔNC (both N- and C-terminus deleted), were constructed and purified. 3α-HSD/CR expression was measured by ELISA to detect the function of HsdR. Functional and biochemical analyses of wild type HsdR and its truncated forms indicated that HsdR may exist as an oligomer where the central domain is crucial for its oligomerization. Gel filtration chromatography revealed that there are two dominant oligomer forms which may be octamers and hexamers. According to electrophoretic mobility shift assays, HsdR specifically binds to its own promoter, where it negatively regulates its own expression. Therefore, the expression of non-functional HsdR variants (an hsdR-gfp fusion mutant and a hsdR gene disrupted mutant) increased compared to the wild type strain, because autorepression of HsdR was prevented. As a consequence, 3α-HSD/CR expression in these hsdR mutant strains was impaired. Combined, in our study we provide evidence that the transcription factor HsdR is a component of the steroid degradation machinery in C. testosteroni, which is active as an oligomer and negatively regulates its own expression.
“3α-羟甾脱氢酶/羰基还原酶调节剂”（HsdR）来源于粪产碱杆菌（Comamonas testosteroni），被鉴定为 LysR 型转录调控因子（LTTR）家族的成员。我们之前已经表明，HsdR 激活编码 3α-羟甾脱氢酶/羰基还原酶（3α-HSD/CR）的 hsdA 基因的表达，该酶是参与甾体化合物降解的重要酶。系统发育分析表明，HsdR 与脑膜炎奈瑟菌的接触调控基因 A（CrgA）有关，后者以同源八聚体的形式存在。因此，为了进一步阐明 HsdR 的调控机制，本研究中我们研究了该转录因子的寡聚状态和自身调控。为了鉴定 HsdR 的活性结构域，构建并纯化了三种截断形式的 HsdRΔN（N 端缺失）、HsdRΔC（C 端缺失）和 HsdRΔNC（N 和 C 端均缺失）。通过 ELISA 检测 3α-HSD/CR 的表达来检测 HsdR 的功能。野生型 HsdR 及其截断形式的功能和生化分析表明，HsdR 可能作为一种寡聚体存在，其中中央结构域对于其寡聚化至关重要。凝胶过滤色谱显示，可能存在两种主要的寡聚体形式，可能是八聚体和六聚体。根据电泳迁移率变动分析，HsdR 特异性地结合其自身的启动子，从而负调控其自身的表达。因此，与野生型菌株相比，非功能 HsdR 变体（hsdR-gfp 融合突变体和 hsdR 基因缺失突变体）的表达增加，因为 HsdR 的自身抑制被阻止。结果，这些 hsdR 突变株中 3α-HSD/CR 的表达受损。总之，本研究提供了证据表明，转录因子 HsdR 是粪产碱杆菌甾体降解机制的组成部分，它作为一种寡聚体发挥作用并负调控其自身表达。
