Chimeric triterpene synthase. A possible model for multifunctional triterpene synthase

Two triterpene synthases, beta-amyrin synthase (EC 5.4.99.-) from Panax ginseng and lupeol synthase (EC 5.4.99.-) from Arabidopsis thaliana, were used to construct a series of chimeric proteins between these two enzymes in order to investigate the region Important for product specificity. Functional expression in yeast and analysis of the synthase products have revealed that chimera 1, in which the N-terminal half is beta-amyrin synthase and the C-terminal half is lupeol synthase, produced both beta-amyrin and lupeol in a 3:1 ratio. By dividing the whole sequence into four regions, all the possible combinations of the two synthases were constructed. Among them, chimera 7, in which only region B (the second quarter from the N-terminus) is beta-amyrin synthase, produced beta-amyrin and lupeol in a 4:1 ratio, indicating the importance of region B in beta-amyrin formation. Another chimera, which was created by the mixed PCR method, produced beta-amyrin and lupeol in a 1:4 ratio, indicating that the sequence which is important for product distribution lies within 80 amino acid residues in region B. The incorporation experiment of [1,2-C-13(2)]acetate showed that, during the formation of lupeol, the final proton abstraction takes place from either of the two gem-dimethyl groups in a 1:1 ratio. This is the first demonstration of the scrambling of methyl groups during the biosynthesis of any terpenoids. On the other hand, no scrambling of methyl groups was observed during beta-amyrin formation, indicating that the isopropyl group of the lupenyl cation must be held tightly by beta-amyrin synthase protein.