Zhang, Yixi; (2018) Structure-based Enzyme Engineering of Glycosyltransferases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Plant UDP-dependent glycosyltransferases (UGTs) play important roles in biology via the glycosylation of secondary metabolites. The future prospects of UGTs look promising, for example, it may serve as promising path for progress in expanding drug targets and synthesising glycan-based drug with enhanced bioactivity. Nevertheless, the current poor understanding of UGTs at molecular level (e.g. kinetic and structure) has led to a limited understanding of their biological roles and has also hampered their potential applications. Aims: This project aims to 1) build up a mass spectrometry (MS) based approach to study families of UGTs including their substrate specificities, kinetic parameters and mechanisms of action (Chapter 3); 2) identify catalytic key amino acids (ckAAs) in the various UGTs (Chapter 4); and finally, 3) apply the methods above in the study of selected Rhamnosyltransferases (RhaTs) 78D1 and 89C1 (Chapter 5). Methodology: A triple quadrupole MS (QQQ-MS) was used as this instrument required limited modification of substrates and provided direct monitoring of the glycosylated product. ‘Full scan mode’ gave the initial screening of any potential glycosylated product, and the ‘product ion’ mode provided additional confirmation of the formation of the glycosylated product. The ‘multiple reaction monitoring (MRM)’ mode quantified the products formation as a function of reaction time and provided kinetic data of the UGTs (Chapter 3). The study of catalytic key amino acids (ckAAs) was based on the multiple sequence alignment (MSA) method via the AA sequence comparison with template UGTs that have known crystal structures. Subsequent site-directed mutagenesis (SDM) was used to substantiate/disprove the functional role of potential ckAAs: mutants (with potential ckAAs mutated) were checked by MS to find out whether the original activities were maintained and/or new activities were gained. A further activity comparison (kcat/KM) between the active mutant and the wild type (WT) could indicate the influence from a particular AA (Chapter 4). Results and conclusions: 29 recombinant UGTs from groups B, D, F, H and L were examined. New donor activities (e.g. UDP-GlcNAc towards 73B4 and 78D2) were reported. Full kinetic studies of these WT UGTs indicated that they followed the Bi-Bi sequential mechanism. Whilst most followed the Bi-Bi random sequential mechanism, exceptions could be found such as that for 73B4 facilitating the UDP-GlcNAc reaction (Chapter 3). Based on the interactions between the donor and template UGT (e.g. VvGT1, PDB 2C1Z), mutations of the potential ckAAs oriented towards the sugar (positions C2, C3, C4 and C6), phosphate and uridine were designed with alanine and an AA with a similar structural and chemical character. An activity comparison (kcat/KM) between the WT and active mutants indicated that most of the potential ckAAs within the PSPG motif inferred from MSA, were conserved and possibly followed a similar interaction pattern. However, exceptions could be found (e.g. 78D2 D380). Taking the results of both MSA and SDM together, the ckAAs in the active sites in each target UGT towards a specific donor were identified (Chapters 4 and 5). Additionally, the study of Rhamnosyltransferases (RhaT) 78D1 and 89C1 showed that new activities were acquired by point mutagenesis: 78D1 N375Q acquired UDP-Glc and UDP-GlcNAc activities; and 89C1 H357Q acquired UDP-Glc activity (Chapter 5).

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