Portsteffen A, Dräger B, Nahrstedt A
Institut für Pharmazeutische Biologie und Phytochemie, Westfälische Wilhelms-Universität, Münster, Germany.
Phytochemistry. 1994 Sep;37(2):391-400. doi: 10.1016/0031-9422(94)85066-6.
In tropane-alkaloid producing plants and root cultures, the reduction of tropinone is a branch-point in secondary metabolism. Two different reductases stereospecifically form the isomeric alcohols tropine (tropan-3 alpha-ol) and pseudotropine (tropan-3 beta-ol). We describe here the purification and characterization of both reductases from transformed root cultures of Datura stramonium. The tropine-forming reductase (TR I, EC 1.1.1.206) was purified 108-fold, the pseudotropine-forming enzyme (TR II, EC 1.1.1.236) was purified 3410-fold to homogeneity. The native molecular weights, both determined by gel chromatography, were 50,700 (TR I) and 77,700 (TR II). In SDS gel electrophoresis a subunit with an M(r) of 27,700 could be identified for TR II. Isoelectric points are at 5.2 (TR I) and 5.7 (TR II). Km values for the physiological substrate tropinone are 1.30 mM (TR I) and 0.11 mM (TR II). NADPH as a cosubstrate shows Km values of 58 microM (TR I) and 16 microM (TR II). NADH is not accepted by either enzyme. The reverse reaction (i.e. oxidation of the alcohol to tropinone) was found only for TR I with a Km of 180 microM. From a detailed analysis of the catalytic activities of TR I and TR II with a range of substrate analogues some key features of the mechanism of reaction can be proposed. The catalytic properties of TR I and TR II are compared with each other and with TR I and TR II activities from other solanaceous species from which these enzymes have been described.
在产生托烷生物碱的植物和根培养物中，托品酮的还原是次生代谢中的一个分支点。两种不同的还原酶立体特异性地形成异构体醇托品（托烷 - 3α - 醇）和假托品（托烷 - 3β - 醇）。我们在此描述了从曼陀罗转化根培养物中纯化和鉴定这两种还原酶的过程。形成托品的还原酶（TR I，EC 1.1.1.206）纯化了108倍，形成假托品的酶（TR II，EC 1.1.1.236）纯化了3410倍达到均一性。通过凝胶色谱法测定的天然分子量分别为50,700（TR I）和77,700（TR II）。在SDS凝胶电泳中，可鉴定出TR II的一个分子量为27,700的亚基。等电点分别为5.2（TR I）和5.7（TR II）。生理底物托品酮的Km值分别为1.30 mM（TR I）和0.11 mM（TR II）。作为共底物的NADPH的Km值分别为58 microM（TR I）和16 microM（TR II）。两种酶均不接受NADH。仅发现TR I存在逆向反应（即将醇氧化为托品酮），其Km为180 microM。通过对TR I和TR II与一系列底物类似物的催化活性进行详细分析，可以提出反应机制的一些关键特征。将TR I和TR II的催化特性相互比较，并与已描述这些酶的其他茄科物种的TR I和TR II活性进行比较。
