Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Abstract

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. Yet such efforts are rare and focus only on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report the first multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio-and stereoselective hydroxylation of a steroid. We developed a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative sign 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 beta-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 lessons for engineering P450s.

Pairing two growth-based, high-throughput selections to fine tune conformational dynamics in oxygenase engineering

“…MD simulations highlight that most of the mutations appear play a role in fine tuning the conformational dynamics of enzyme and cofactor, which are subtle effects that are challenging to design without a high-throughput method. Conformational dynamics is particularly critical for catalysis in oxygenases [20][21][22][23] . We envision the wide application of our selections in directed evolution of these complex and powerful enzymes.…”

“…Oxygenases are promising catalysts, but difficult to engineer due to their highly complex behavior in sampling multiple conformational states during catalysis [20][21][22][23] . We envision that the tools developed here will make the conformational dynamics a more tunable element in oxygenase engineering.…”

A Pioneering Career in Catalysis: Manfred T. Reetz

“…Full deconvolution by also generating and testing single mutants M16A and L17F as well as all double combinations would have provided even more information on possible epistatic effects. Indeed, as already indicated above, the Reetz group pioneered the use of complete deconvolution as a method for uncovering cooperative, neutral, and antagonistic mutational effects on stereoselectivity at all evolutionary stages in terms of ΔΔG ‡ …”

“…In fact, they reveal nothing about mutational additivity or nonadditivity. Extensive information on this unique branch of directed evolution is available, featuring the details of evolutionary stereoselectivity pathways in terms of ΔΔ G ‡ values. ,,,, …”