The reduction of tropinone in Datura stramonium root cultures by two specific reductases

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.