Identifying functional domains within terpene cyclases using a domain-swapping strategy

Cyclic terpenes and terpenoids are found throughout nature, They comprise an especially important class of compounds from plants that mediate plant-environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities, Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C-10, C-15, and C-20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exon position and size within the genes, Functional domains responsible for a terminal enzymatic step were identified by swapping regions approximating exons between a Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) gene and a Hyoscyamus muticus vetispiradiene synthase (HVS) gene and by characterization of the resulting chimeric enzymes expressed in bacteria. While exon 4 of the TEAS gene conferred specificity for the predominant reaction products of the tobacco enzyme, exon 6 of the HVS gene conferred specificity for the predominant reaction product(s) of the Hyoscyamus enzyme, Combining these two functional domains of the TEAS and HVS genes resulted in a novel enzyme capable of synthesizing reaction products reflective of both parent enzymes. The relative ratio of the TEAS and HVS reaction products was also influenced by the source of exon 5 present in the new chimeric enzymes, The association of catalytic activities with conserved but separate exonic domains suggests a general means for generating additional novel terpene cyclases.