Screening Glycosyltransferases for Enzymatic Activity

Plant …, 2004

PLANT PHYSIOLOGY, 2010

The Plant Journal, 1999

Current Opinion in Plant Biology, 2001

Glycoconjugate Journal, 2010

Glycosyltransferases (GTs) are a group of enzymes that transfer sugar moieties from active donors e.g uridine diphosphate (UDP) glucose to acceptors which are very important to living organisms. Addition of sugars to compounds play important role in the detoxification, solubility, reactivity and stability of compounds. However, the substrate specificity and mechanism of GTs are not fully understood. This work aims to assess the function of specific amino acids in UGTs from Arabidopsis thaliana 76E3, 76E4, 76E5, 76C2, 76C4 and 76D1. Site directed mutagenesis was performed to make mutants whose amino acids has been changed at assumed active sites T141, D374 and Q375 to alanine. These sites were chosen because they were indicated in the binding of sugar donor hydroxyl groups shown in the crystal structure of VvGT1. The mutants made were UGT76E4-T135A, UGT76E4-E374A, UGT76E4-Q375A, UGT76C4-T135A, UGT76C4-W374A, UGT76C4-D373A. Others are UGT76E5-T134A, UGT76E5-Q366A, UGT76E3-T131A, UGT76E3-Q370A, UGT76C2-Q375A and UGT76D1-Q371A. Mass spectrometry was used to test activities of the mutant enzymes. All the 12 mutants showed loss of activity with UDP-glucose as sugar donor after mutagenesis in the mass spectrums. Complete loss of activity clearly suggests that peptides present at the various sites of mutation are highly important to the enzymes function inglucosylating compounds. This work further affirms that glycosyltransferases can be modified using site directed mutagenesis which could be of high importance in cancer drug development. Keywords: UDP glucose, Glycosyltransferases, Site directed mutagenesis, mass spectrometry

Plant and Cell Physiology, 2018

PhD Thesis, 2020

Background: Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promising potential as drug targets as well as catalysts in the synthesis of glycosides of medicinal importance. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. For example, only two crystal structures of Arabidopsis thaliana UGTs are currently solved of the 122 genes available. In addition, more than half of these UGTs are yet to be biochemically characterised. Aims: This research aims to i) investigate qualitatively substrate specificities of Arabidopsis thaliana UGTs from selected families using mass spectrometry (MS) based methods and to study the kinetic parameters via bioluminescence (Chapter 3); ii) produce and study homology models of the UGTs (novel) to further understand their substrate preferences and key catalytic amino acid residues involved (Chapter 4); and lastly iii) manipulate rationally the active sites of UGTs to engineer mutant UGTs of improved donor substrate activity (Chapter 5). Methodology: Direct monitoring of products of glycosylation was done using triple quadrupole mass spectrometry (QQQ-MS) as it involves limited substrate modification. Full scan and product ion screening modes identifies the potential glycosylated product and confirms the product formation respectively. The kinetic data of the UGTs was determined via UDP-Glo glycosyltransferase assay which measured the amount of UDP released as a function of time (Chapter 3). Homology modeling was employed in the absence of experimental crystal structures to identify structural differences in these UGTs which drive substrate preferences. Docking of ligand substrates into the model UGTs was done to understand interactions at the molecular level (Chapter 4). Site directed mutagenesis was used to produce mutant UGTs to substantiate the functional roles of potential key amino acids. These mutations were rationally (sequence-based and structure-based) designed (Chapter 5). Results and conclusions: 22 recombinant UGTs from groups L, H and D were selected for substrate screening. 15 of these were successfully expressed while 8 UGTs show glycosylation activity. 76E1 displayed the highest acceptor substrate recognition while both 76E5 and 76E1 showed highest donor recognition. Very low Km at μM scale suggests enzymes good affinity for the donor substrates with 76E5 showing stronger preference for UDP-Gal and UDP-GlcNAc (Chapter 3). Homology models of five group H UGTs were constructed, validated and substrate ligands docked into them. With a focus on donor sugar interactions, key amino acid residues interacting at specific positions of each model UGT were shown. In addition, a major structural difference in N3/Nα3 region of 76E1 was found which may be responsible for its higher acceptor substrate recognition (Chapter 4). 4 The usefulness and predictive power of these models helped design mutant UGTs. Rationally designed mutant UGTs such as 76E2 N320S, 76E4 K275L, 76D1 P129T and 76E2 D374E displayed improved substrate recognition which also highlights the functional roles of those amino acid residues (Chapter 5).

Plant Molecular Biology, 2004

Biochimica Et Biophysica Acta-general Subjects, 1999