Protein Evolution and Emergence of Novel Catalytic Functions

Enzyme evolution underlies major expansions of metabolic complexity with profound biological implications. In this presentation, I will discuss emergence of cyclization reactions catalyzed by terpene synthases. Cyclic terpenes mediate numerous biological functions in modern plants (such as attracting pollinators and repelling microbial pathogens), and provide bioactive compounds for human use, including artemisinin, the most effective treatment for malaria currently available. Guided by the available structural, kinetic, and sequence data, we have constructed mutant libraries which include combinations of amino acids responsible for inducing cyclization reactions in an enzyme that produces beta-farnesene, a linear hydrocarbon chain. We have used measurements of kinetic rates and mass spectrometry in order to assess catalytic efficiency and specificity of the mutant enzymes. Using spin glass models adapted from statistical physics, we have inferred evolutionary patterns of enzyme energetics, as well as sequence signatures of active vs. inactive terpene synthases. Our studies provide quantitative insights into evolutionary dynamics of a major enzyme family, and highlight the importance of epistasis in molecular evolution.