Tailoring Enzyme Active Sites using an Expanded Genetic Code

Abstract

Enzyme design and engineering strategies typically rely on nature's alphabet of twenty canonical amino acids, which contain only limited range of functional groups. This limited functionality restricts our ability to precisely modulate protein structure and function. To address these limitations, powerful genetic code expansion methods have been developed that allow hundreds of structurally diverse amino acids to be introduced into proteins in a site-selective manner. In this thesis, we highlight how an expanded alphabet of amino acids can be used as tools to probe complex enzyme mechanisms, augment biocatalyst function or to embed entirely new modes of catalysis into proteins. In Chapter 3, we exploit a close structural analogue of tryptophan to understand how hydrogen bonding interactions with high-energy ferryl heme intermediates influences their structure and reactivity. In Chapter 4, we examine the influence of axial ligand strength on the reactivity of ferryl intermediates in a non-heme iron oxygenase, by comparing the C-H/C-D abstraction kinetics in histidine and Nδ-methyl-histidine ligated VioC. Finally, in Chapter 5, we develop an efficient and enantioselective photoenzyme for intra- and intermolecular [2+2]-cycloadditions by embedding a benzophenone triplet photosensitiser into the hydrophobic core of a designed Diels-Alderase. Taken together, these studies demonstrate the power of genetic code expansion technology in enzyme design and engineering research.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anthony Green (Supervisor) & Sarah Lovelock (Supervisor)