Exploring the emergence of pesticide degradation in the α/β hydrolase superfamily
type: CreativeWork
description: How do new enzymatic motifs emerge in evolution? Charting new mechanistic solutions for biocatalytic challenges is a core obstacle in the constant evolutionary adaptation of living organisms. If understood and harnessed, these processes hold the potential to revolutionise industrial processes in the chemical and pharmaceutical industry. The recent massive release of new, man-made substance classes into the environment in the form of pesticides provides a dynamic testing ground to observe protein evolution in fast-forward mode. In this global experiment, we can observe the emergence of new enzymatic activities and compare the adaptability of different catalytic motifs. Among the xenobiotic substances used, phosphotriester pesticides range among the most toxic. Phosphotriester pesticides were designed to irreversibly inhibit the catalytic triad of synaptic acetylcholinesterase. Numerous enzymes from different evolutionary origins have rapidly evolved to hydrolyse phosphotriesters since. Interestingly, all known enzymes have convergently reached the same mechanistic solution by recruiting divalent cations as a cofactor for metal-ion catalysis. The starting point of this study is the α/β hydrolase P91. This enzyme was recently identified in a functional metagenomic screening and displays weak promiscuous phosphotriesterase (PTE) activity. P91’s PTE activity is remarkable because, in contrast to other currently known PTEs, it utilises a fundamentally different, metal-independent mechanism. At the same time, it has the same fold and a very similar active site configuration as acetylcholinesterase, the biochemical target of most organophosphate pesticides and nerve gases. In this dissertation, I subject P91 to directed evolution in order to explore the evolvability of its catalytic motif to this new enzymatic activity (chapter 2). To be able to cover otherwise inaccessible sequence space, I use droplet microfluidics to screen large libraries of enzyme variants in high thro…
abstract: How do new enzymatic motifs emerge in evolution? Charting new mechanistic solutions for biocatalytic challenges is a core obstacle in the constant evolutionary adaptation of living organisms. If understood and harnessed, these processes hold the potential to revolutionise industrial processes in the chemical and pharmaceutical industry. The recent massive release of new, man-made substance classes into the environment in the form of pesticides provides a dynamic testing ground to observe protein evolution in fast-forward mode. In this global experiment, we can observe the emergence of new enzymatic activities and compare the adaptability of different catalytic motifs. Among the xenobiotic substances used, phosphotriester pesticides range among the most toxic. Phosphotriester pesticides were designed to irreversibly inhibit the catalytic triad of synaptic acetylcholinesterase. Numerous enzymes from different evolutionary origins have rapidly evolved to hydrolyse phosphotriesters since. Interestingly, all known enzymes have convergently reached the same mechanistic solution by recruiting divalent cations as a cofactor for metal-ion catalysis. The starting point of this study is the α/β hydrolase P91. This enzyme was recently identified in a functional metagenomic screening and displays weak promiscuous phosphotriesterase (PTE) activity. P91’s PTE activity is remarkable because, in contrast to other currently known PTEs, it utilises a fundamentally different, metal-independent mechanism. At the same time, it has the same fold and a very similar active site configuration as acetylcholinesterase, the biochemical target of most organophosphate pesticides and nerve gases. In this dissertation, I subject P91 to directed evolution in order to explore the evolvability of its catalytic motif to this new enzymatic activity (chapter 2). To be able to cover otherwise inaccessible sequence space, I use droplet microfluidics to screen large libraries of enzyme variants in high thro…
author: Schnettler Fernández, Juan David F
language: eng
keywords: protein engineering, directed evolution, high-throughput screening, microfluidics, phosphotriesterase, enzyme promiscuity, catalytic mechanism
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2021-05-05
date modified: 2026-07-22T22:24:28Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.83346