Laboratory of
Computational Biochemistry

Selected for Oral Presentation:

From Molecular Phylogenetics to Quantum Chemistry:
Discovering How Sesquiterpene Synthases Evolve New Functions

Authors: Troy Wymore (NRBSC) and Charles L. Brooks III (University of Michigan)

The attainment of new catalytic functions from an existing protein scaffold is a major force guiding evolutionary change but one that is perhaps only beginning to be understood. Understanding the evolution of enzymatic function at the physicochemical level requires first that probable evolutionary paths that interconvert an enzyme’s specific function from one to another through accessible mutational changes be discovered and thus a catalytic landscape be defined. Through a landmark study of sesquiterpene synthases in which a 5-epi-aristolocholene synthase was transformed to a premnaspirodiene synthase through mutational swaps of nine residues (none of which make specific contacts with the substrate) as well as experimental classification of all 512 proteins with different combinations of these nine residues a catalytic landscape underlying the evolution of sesquiterpene chemical diversity was revealed [O’Maille et al, Nature Chemical Biology, 2008]. Also of significance, the catalytic cycle of both synthases passes through several common intermediate states and only diverge in the last few chemical reactions. Because of all these interesting properties, elucidating the mechanistic basis for this evolution of new function has broad implications for a more complete understanding of allostery, functional epistasis, evolvability and protein design concepts.

In this presentation, we will describe our integrated approach that employs molecular phylogenetic analysis, atomistic molecular dynamics simulations of the solvated enzyme with an intermediate state and quantum chemical calculations that together begins to reveal a mechanism by which sesquiterpene synthases evolve novel functions. Evolving a new enzyme function by mutation of active site residues can often have negative effects on protein stability and thus compensating mutations are often necessary. In addition, it has been shown that “non-functional” outside-the-active-site residue(s) must be mutated first before a “functional” active site residue can be mutated. This context dependence, or epistasis, restricts the possible evolutionary pathways to novel functions while also revealing the interdependent network of interactions in the protein. Our phylogenetic analysis and molecular modeling results support the conclusion that plant sesquiterpene synthases evolve new functions in two ways that would have a minimal impact on protein stability; 1) swapping a hydrophobic residue in the active site with another hydrophobic one could have a large impact on the binding mode of the farnesyl substrate and the fate of intermolecular reactions and 2) substituting residues outside the active site but still adjacent to a highly conserved Tyr-520-Asp-525 dyad (identified through our analysis) would shift the dyads’ position relative to the farnesyl substrate resulting in proton transfers from different locations on the substrate. The important role of proton transfer in generating product diversity has not been highlighted by previous studies of sesquiterpene synthases.