Understanding Structure-Function Relationships in a Sesquiterpene Synthase family through the Examination of Natural and Unnatural Variants

Terpenes are one of the largest groups of plant secondary metabolites that are utilized for a multitude of purposes from defense to pollinator attraction in plants, and medicinal purposes in humans. The synthesis of the extensive array of terpenoid compounds is complex and nuanced; hence the following work attempts to elucidate one portion of the terpene story, focusing on 5-epi- aristolchene synthase (EAS). The terpene synthase EAS is a member of a simple three-enzyme sesquiterpene biosynthetic pathway that ultimately produces the hydroxylated sesquiterpene phytoalexin, capsidiol. Here we begin the investigation of the natural variation found in EAS-like enzymes and their associated biochemical properties, across the ecologically diverse Nicotiana plant genus. The work entails an in-depth examination of functional diversification of an enzyme family critical to pathogen resistance in plants, where EAS-like genes were cloned, sequenced, and expressed recombinantly from several species of the Nicotiana genus of plants and the resultant enzymes were biochemically characterized. Characterization focused on three fundamental properties of enzymes, namely thermostability, kinetic efficiency, and product identity and diversity. From the aforementioned survey, two EAS- like enzyme variants were isolated that produced distinct product profiles when compared to the relatively well- characterized Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The in-depth study of these two EAS-like synthases illuminates several factors that influence the distribution and identity of the products that are synthesized by these enzyme