Kristan Katja, Brunskole Mojca, Stojan Jure, Rizner Tea Lanisnik
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Chem Biol Interact. 2009 Mar 16;178(1-3):295-302. doi: 10.1016/j.cbi.2008.09.032. Epub 2008 Oct 9.
Two homologous fungal short-chain dehydrogenase/reductase (SDR) proteins have been cloned from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus) and expressed in Escherichia coli: trihydroxynaphthalene reductase (3HNR), an enzyme of the melanin biosynthetic pathway that catalyzes the conversion of 1,3,8-trihydroxynaphthalene to vermelone, and 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl), which acts on androgens and estrogens, although its physiological substrate remains to be defined. In the present study, we have compared the structures, specificities to substrates and inhibitors, temperature and pH optima of 3HNR and 17beta-HSDcl. Sequence analysis and homology-built models revealed that these enzymes are highly similar. Both of these enzymes are NADP(H)-preferring reductases and act on steroids at position 17; however, 17beta-HSDcl presented considerably higher initial rates than 3HNR. In vitro, 17beta-HSDcl preferably catalyzed the reduction of 4-estrene-3,17-dione, while the best steroid substrate for 3HNR was 5alpha-androstane-3,17-dione. On the other hand, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), an artificial substrate of 3HNR, was oxidized rapidly by 3HNR, while it was not a substrate for 17beta-HSDcl. Additionally, our data show that tricyclazole, a specific inhibitor of 3HNR, is 100-fold less effective for 17beta-HSDcl inhibition, while flavonoids can inhibit both 3HNR and 17beta-HSDcl. We have also examined the effects of temperature and pH on the oxidation of DDBO by 3HNR and the oxidation of 4-estrene-17beta-ol-3-one by 17beta-HSDcl. The apparent optimal temperature for 3HNR activity was between 25 and 30 degrees C, while it was between 40 and 45 degrees C for 17beta-HSDcl activity. The pH optimum of 3HNR activity was between 8 and 9, and for 17beta-HSDcl, between 7 and 8. Our data show that in spite of high homology and similar backbone structure, differences between 3HNR and 17beta-HSDcl were not only in substrate specificities, but also in temperature and pH optima.
从新月弯孢霉（有性型：月状旋孢腔菌）中克隆出了两种同源的真菌短链脱氢酶/还原酶（SDR）蛋白，并在大肠杆菌中进行了表达：三羟基萘还原酶（3HNR），它是黑色素生物合成途径中的一种酶，催化1,3,8 - 三羟基萘转化为紫红素；以及17β - 羟基类固醇脱氢酶（17β - HSDcl），它作用于雄激素和雌激素，不过其生理底物仍有待确定。在本研究中，我们比较了3HNR和17β - HSDcl的结构、对底物和抑制剂的特异性、最适温度和最适pH值。序列分析和同源构建模型显示这些酶高度相似。这两种酶都是偏好NADP(H)的还原酶，且都作用于17位的类固醇；然而，17β - HSDcl的初始反应速率比3HNR高得多。在体外，17β - HSDcl更倾向于催化4 - 雌烯 - 3,17 - 二酮的还原，而3HNR的最佳类固醇底物是5α - 雄烷 - 3,17 - 二酮。另一方面，3HNR的人工底物2,3 - 二氢 - 2,5 - 二羟基 - 4H - 苯并吡喃 - 4 - 酮（DDBO）能被3HNR快速氧化，而它不是17β - HSDcl的底物。此外，我们的数据表明，3HNR的特异性抑制剂三环唑对17β - HSDcl的抑制效果要低100倍，而黄酮类化合物能同时抑制3HNR和17β - HSDcl。我们还研究了温度和pH值对3HNR催化DDBO氧化以及17β - HSDcl催化4 - 雌烯 - 17β - 醇 - 3 - 酮氧化的影响。3HNR活性的表观最适温度在25至30摄氏度之间，而17β - HSDcl活性的表观最适温度在40至45摄氏度之间。3HNR活性的最适pH值在8至9之间，17β - HSDcl的最适pH值在7至8之间。我们的数据表明，尽管3HNR和17β - HSDcl具有高度同源性和相似的主链结构，但它们之间的差异不仅体现在底物特异性上，还体现在最适温度和最适pH值上。
