Abstract
Terpenes form the largest class of natural products, providing a wide variety of uses with applications ranging from fragrances to therapeutics, to even serving as potential biofuels. Pentalenene synthase (PS), a sesquiterpene cyclase, is known to make pentalenene via an anti-Markovnikov mechanism. In order to understand how PS proceeds along this seemingly less favorable mechanism, in this thesis, we explore the crystal structure of PS in complex with the substrate analog 12,13-difluorofarnesyl pyrophosphate (12,13-DFFPP). From our structure, we observed a hydrophobic region responsible for coordinating the carbon chain of the substrate. Within the non-polar region, we found residue F76 to stabilize a carbocation at C9 of the chain by means of a cation-p interaction. We believe this helps make the anti-Markovnikov mechanism more
favorable. In order to confirm our hypothesis, we made two mutants, PS F76Y and F76W. F76Y displayed activity indistinguishable from the wild-type enzyme. In contrast, F76W produced less pentalenene and 20% of a new product, a-humulene, which we reason to be because the pyrrole ring within the tryptophan sidechain is stabilizing the carbocation at position C10. We crystallized the mutant protein, PS F76W, and modeled in 12,13-DFFPP. We believe the pyrrole ring within the sidechain is positioned to stabilize the carbocation at C10 and the hexanes ring positioned to interact with the carbocation at C9, resulting in a mixture of products. Ultimately, the unfavorable antiMarkovnikov mechanism is stabilized through cation-p interactions by aromatic residues within the active site, allowing for specific sesquiterpene products to be made depending on the positioning of the residue .