Chan, Shu Ann
Description
The demand for O-glucuronides as potential therapeutic products
and biomarkers continue to increase. However, large-scale
synthesis of O glucuronides remains a challenge for the industry,
and has prompted the development of an alternative synthetic
routes. This dissertation extends from a previous enzyme
engineering work that had introduced a site-specific mutation
E504G in β glucuronidase (β-GUS), resulting in a functional
glucuronylsynthase (Syn). However,...[Show more] the synthetic activity of Syn
is low and leaves ample scope for improvement. The work described
in this thesis aims to produce a more efficient
glucuronylsynthase using different enzyme engineering approaches.
Two separate strategies were employed to achieve our objective.
The first strategy engages a two-step process where the β-GUS is
first engineered to have higher activity in the presence of
excess substrate; 10–20 times its Km. This is followed by
site-specific mutation E504G to convert the β-GUS variant into a
Syn. The second strategy engineers the glucuronylsynthase
directly. Chapter 3 describes the attempt to improve the activity
of the native enzyme in the presence of t-BuOH, a solvent that
was found to improve the chemistry of the glucuronylsynthase
chemoenzymatic reaction. The engineering attempt produced a
potential variant with a mutation at its C-terminal region,
L561S, that is more active in the presence of the solvent. This
mutation appears to be a determinant mutation. Biophysical
characterization of the enzyme revealed that this improvement is
not due to increased stability in t-BuOH, while our analysis of
the crystal structure suggests that the mutation improved the
activity by increasing loop flexibility at the C-terminal region.
Subsequently, I incorporated E504G into the β-GUS variant, but
this did not translate into a better glucuronylsynthase variant.
Chapter 4 describes the second strategy. Two mutations, H162Q and
Y160G, at the N-terminal region were found to boost the synthetic
activity but this was not accompanied by improvement in their
thermostability nor solvent stability. However, combining the
results from the biophysical characterization experiments and
observations from the structural examination on 3K4D, it can be
inferred that the mutations promote glucuronylsynthase activity
by modulating the active site of the Syn so that it would favour
the glucuronyl donor substrate. Therefore, these mutations would
serve as concrete starting points for further evolution program
of the Syn. Chapter 5 explores the potential reason that could account for
the lack of success in transposing the potency of L561S to the
glucuronylsynthase system. The work here is driven by the
hypothesis that translational misincorporation introduced
contaminating wild-type during enzyme expression. Essentially,
this chapter highlights the potential pitfall of the
glucuronylsynthase system and describes potential strategies to
avoid this pitfall. Finally, Chapter 6 builds upon the results
from β GUS engineering and explores the mutational tolerance of
β GUS. Its mutational tolerance is compared with another enzyme
that is structurally less complex (β-lactamase, TEM-1). In
addition, its mutational tolerance with different substrate is
also compared. This exercise attempts to provide insights into
the elements that would drive the adaption process of the β-GUS.
Consequently, we expect this study to facilitate future directed
evolution studies of the β-GUS and the glucuronylsynthase.
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