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.[1]References
- The reduction of tropinone in Datura stramonium root cultures by two specific reductases. Portsteffen, A., Dräger, B., Nahrstedt, A. Phytochemistry (1994) [Pubmed]
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