CLONING AND BACTERIAL EXPRESSION OF A SESQUITERPENE CYCLASE FROM HYOSCYAMUS-MUTICUS AND ITS MOLECULAR COMPARISON TO RELATED TERPENE CYCLASES

Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene c yclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional c loning procedures, RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elic itor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated th at each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyc lase) and casbene synthase (castor bean diterpene cyclase), and the ve tispiradiene synthase amino acid sequence was 77% identical with and 8 1% similar to the tobacco sesquiterpene cyclase, Regions of the vetisp iradiene synthase sequence centered around amino acids 60, 100, and 37 0 were conspicuously different relative to the tobacco sesquiterpene c yclase, The sequence similarity between the tobacco and H. muticus enz ymes is suggested to be reflective of the conservation of several part ial reactions common to both enzymes, and the differences may be refle ctive of a partial reaction unique to each enzyme.