Structural and Functional Studies of the Natural Diels-Alderase AbyU and the Bacillus Subtilis Enzyme TrpE

Abstract

The discovery and development of natural Diels-Alderases has become an increasingly fertile
area of research over the past decades. Interest in these biocatalysts has been motivated
by a desire to exploit their ability to install substituted cyclohexenes into a diverse array
of bioactive chemical scaffolds including drugs and agrochemicals. Although the existence of
naturally evolved Diels-Alderases has long been a topic of some dispute, recent studies have
resulted in the identification and characterisation of a number of enzymes capable of catalysing
this reaction. As a consequence, attention is now turning to exploring the substrate selectivities of
these protein catalysts, along with investigations into their potential for engineering, to enable the
targeted biosynthesis of non-natural compounds. This thesis outlines progress in the expression,
purification and characterisation of point mutants of the natural stand-alone Diels-Alderase,
AbyU. Mechanistic details of the AbyU catalysed reaction have been investigated, employing
structural and kinetic methods, in tandem with a mutagenesis programme. Studies have also
explored the capacity of WT AbyU and selected mutants to accept and act upon non-cognate
substrates, establishing the utility of natural Diels-Alderases for use in the targeted synthesis of
abyssologue antibiotics.
In complimentary studies, the tryptophan biosynthetic enzyme TrpE, and its homologue PabB,
from the folate biosynthetic pathway, have been subjected to structural and functional analysis.
Both enzymes have been produced in recombinant form and subjected to crystallisation screening.
In vitro enzyme assays, together with modelling studies, have established the molecular basis
of regioselectivity in these two enzymes and provided a framework for understanding their
inhibition by antimicrobial natural products. These studies will aid in the development of
synthetic inhibitors of these important enzymes.

Date of Award	4 Feb 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Paul R Race (Supervisor)