Engineering the glycoside hydrolase β-glucuronidase (β-GUS) to improve a non-native activity for the synthesis of O-glucuronides
Creators: name: Chan, Shu Ann
Publisher: name: The Australian National University
Year: 2016
Subjects: Glucuronidase
glucuronylsynthase
O-glucuronide
translational misincorporation
mutational tolerance

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, 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.