PURIFICATION AND CHARACTERIZATION OF SOLANIDINE GLUCOSYLTRANSFERASE FROM THE POTATO (SOLANUM-TUBEROSUM)

The glycosylations of the spirostanol alkaloid solanidine in potatoes are considered to be the terminal steps in the synthesis of the potentially toxic glycoalkaloids alpha-solanine and alpha-chaconine. To better define the biosynthetic pathways to Solanum alkaloids, we describe an approximately 600-fold purification of the enzyme solanidine UDP-glucose glucosyltransferase (SGT) to near homogeneity from potato sprouts of the cultivar Lemhi. Purification was achieved by a combination of ammonium sulfate precipitation and FPLC anion-exchange, gel filtration, and chromatofocusing chromatography. The isolation of this enzyme was complicated by its copurification with patatin, the primary tuber storage protein. Separation of the two proteins was achieved only by binding the glycosylated patatin to a concanavalin A affinity resin under conditions where the SGT activity did not bind. Analysis of the purified enzyme by SDS/denaturing-PAGE revealed a major protein band of M(r) 38 000. The native molecular weight of SGT determined by gel filtration chromatography was approximately 36 000, indicating that the native enzyme exists as a monomer. The purified enzyme preparation had a pH optimum of pH 6.4-6.8. Kinetic analysis showed K(m) values of 37-mu-M for UDP-glucose and 20-mu-M for solanidine. The closely related spirosolane alkaloids tomatidine and solasodine were glucosylated at rates 2 and 3 times that of solanidine, respectively, whereas 3-beta-hydroxy steroids lacking a ring nitrogen such as cholesterol, diosgenin, digoxigenin, and beta-sitosterol did not serve as glucose acceptors. No enzymic activity was detected when UDP-galactose was used as a substrate, suggesting either that the pathway to alpha-solanine requires a separate galactosyltransferase activity or that gamma-chaconine (glucosylsolanidine) is first converted to gamma-solanine (galactosylsolanidine) by a specific epimerase. The significance of these findings for the control of the biosynthesis of glycoalkaloids at the molecular level in Solanaceous plants is discussed.