Abstract
Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.
Highlights
Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution
The identification of epistasis and its molecular mechanism are crucial for understanding protein function, but these are hardly ever explored in laboratory evolution studies of multiparametric optimization
While such landscapes have revealed that usually many pathways are accessible in laboratory evolution of enzymes as catalysts in organic chemistry[4,7,56,64], different conclusions have been made in evolutionary biology[8,9,10,11]
Summary
Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. Directed evolution has provided important insights into the relationship between protein sequence and function[4,5,6], yet understanding the intricacies of non-additive epistatic effects remains a challenge[7]. Epistatic effects can be negative (antagonistic/deleterious) or positive (synergistic/cooperative) if the respective predictive value is smaller or greater in sign/magnitude than the expected value under additivity. Based on studies of natural and laboratory protein evolution, negative[10] or positive[11] epistasis is more widespread than originally thought[7]
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