Abstract
Terpene synthases perform incredible chemistry to produce complex final products, while the enzyme only takes in a simple achiral substrate. This is done by the donation of 𝜋-bonds from across the substrate’s prenyl chain, generating and rearranging high-energy carbocations to create large fused-ring systems. When cyclizing, terpene synthases typically follow the Markovnikov rule when a 𝜋-bond donates, so the more stable carbocation intermediate forms. However, this is not always the case. Pentalenene synthase (PS), for example, catalyzes an initial anti-Markovnikov cyclization on the C10-C11 olefin of the substrate farnesyl pyrophosphate (FPP). Following a 1,2-hydride shift, the positive charge exists on C9, and through analysis of X-ray crystallography, this charge was found to be stabilized by an aromatic residue (F76) poised right next to C9. With this discovery, we have continued to investigate other enzymes that perform anti-Markovnikov cyclizations. With more enzymes to study, a phylogenetic tree was constructed to categorize enzymes as being related to PS and within its clade. Isohirsut-4-ene synthase (IHS) is an enzyme within the PS clade that also catalyzes an anti-Markovnikov initial cyclization, has an aromatic residue near C9, and also makes a triquinane final product. This project focuses on the mechanism behind PS and IHS, along with other closely related enzymes, and how the final product is formed within the active site, as well as the specific residues within the enzyme that allow for this chemistry to happen. We present these results in the context of PS to show IHS to be an equally powerful enzyme in its chemical prowess to PS.