Abstract
Terpene synthases (TPSs) produce the largest and most diverse class of natural products. They engage a small number of highly reactive substrates and perform complex intramolecular reactions such as anti-Markovnikov cyclizations. Previously our lab has shown that pentalenene synthase (PS) is dependent on F76 aromaticity for its concerted anti-Markovnikov reaction. This thesis addresses evolutionary questions concerning the emergence of anti-Markovnikov specificity for PS and two related TPSs. Using ancestral sequence reconstruction to define the evolutionary history of TPSs, I show that aromaticity was present at position 76 before anti-Markovnikov specificity emerged and that higher order epistasis activated F76 for pentalenene specificity. Using ancestral sequence reconstruction (ASR), I show reverse evolution occurring for the first time in a protein family among other unique evolutionary processes. Additionally, I report the first structures of caryolan-1-ol synthase with substrate analogue bound showing the multistep nature of its anti-Markovnikov mechanism compared to that of PS. Finally, I show that modern and ancestral TPSs can produce complex, polymethylated terpenes through incubation with methylated substrates. Studying the evolution of TPSs using ASR can not only aid in identifying residues and epistasis that control specificity, but also can contribute to molecular diversity by accessing novel products from ancestral sources.