Sparse epistatic patterns in the evolution of terpene synthases

We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes - complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of the emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-β-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon. Each mutant enzyme in our synthetic libraries was characterized biochemically, resulting in reaction rate measurements for 7 cyclic and 4 linear products. We used this reaction rate data as input to the Michaelis-Menten model of enzyme kinetics, in which the free energies were represented as sums of one-amino-acid contributions and two-amino-acid couplings. Our model predicts measured reaction rates with high accuracy and yields free energy landscapes characterized by relatively few coupling terms. As a result, the Michaelis-Menten free energy landscapes have simple, interpretable structure and exhibit little epistasis. We have also developed biophysical fitness models based on the assumption that highly-fit enzymes have evolved to maximize the output of correct products, such as cyclic products or a specific product of interest, while minimizing the input of incorrect products, which may be toxic or costly to remove and degrade. This approach results in a fitness landscape which is a non-linear function of Michaelis-Menten free energies and is therefore considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of sequence determinants and molecular mechanisms underlying evolutionary emergence of novel enzymatic functions in the context of micro-evolutionary exploration of sequence space around wild-type enzymes.