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UBC Theses and Dissertations

The evolution of novel xenobiotic organophosphate activity in the metallo-β-lactamase superfamily Yang, Gloria


New protein functions often evolve through the recruitment and optimization of latent promiscuous activities. How do mutations alter the molecular architecture to change function? The overarching goal of my thesis is to provide answers to this question, utilizing a novel xenobiotic organophosphate hydrolase (OPH) activity as model. Directed evolution performed on an N-acyl homoserine (AHL) lactonase enzyme possessing promiscuous OPH activity demonstrated that the new function can be quickly optimized via a handful of mutations that rearranged active site residues to adapt to the new substrate. Ancestral sequence reconstruction (ASR) conducted on a recently evolved OPH enzyme, methyl-parathion hydrolase (MPH), revealed that the OPH activity emerged from an ancestral lactonase enzyme via five mutations that enlarged the active site to increase complementarity to the new substrate. Subsequent generation of the adaptive fitness landscapes formed by these five mutations uncovered a prevalence of epistatic interactions that constrained the number of accessible evolutionary trajectories. Furthermore, the topologies of the landscapes drastically change in response to subtle differences in substrate substituents. Finally, characterization of several extant lactonase orthologs of MPH revealed that sequence divergence has resulted in lower levels of promiscuous OPH activities in the orthologs compared to the ancestral enzyme that gave rise to MPH. Moreover, the five mutations fail to substantially increase OPH activity in the genetic backgrounds of the orthologs. Comparative directed evolution conducted on the MPH ancestor and the orthologs towards OPH activity show that the ancestral enzyme is able to improve the new function more rapidly. Overall, the results of this thesis contribute to our understanding of enzyme evolution, and will help to better protein engineering and design in the future.

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