Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, ...limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.
Essential oils are volatile constituents that give aromatic plants their characteristic odour. The application of these plant actives in food, agriculture, pharmaceutics, and cosmetics has been ...widely studied. Aromatherapy, a complementary therapy involving the use of essential oils to treat several diseases ranging from microbial infections to metabolic dysfunctions, has been utilised for centuries. Anticancer, antimicrobial, and anti-inflammatory activities are well-established among other pharmacological properties of these aromatic oils. The oils, which are composed mainly of terpene-based compounds, have also been explored as nutraceuticals, alternative green preservatives, and functional additives in foods. However, due to their physicochemical properties, viz high volatility and low aqueous solubility, essential oil delivery to target receptors were challenging when administered as chemotherapeutics. Hence, formulating essential oils with suitable excipients to enhance their delivery and bioavailability, invariably improving their bioactivity and therapeutic efficacy becomes expedient. Nanotechnology presents a unique strategy to develop a particulate delivery system for the controlled, sustained, and extended release of essential oils. In this review, we examine and summarize the trends and developments in the formulation of essential oils using polymeric nanoparticles.
In this report, we cloned and characterised four members of group H glycosyltransferases (GTs) by studying their substrate specificities and kinetics. The formation of products and possible ...glycosylation position was confirmed using MS/MS. The results revealed that 76E1 and 76E5 have broader donor specificity, including UDP-glucose (UDPGlc), UDP-galactose (UDPGal) and UDP-
-acetylglucosamine (UDPGlcNAc) with various flavonoids as acceptor substrates. Pseudo-single substrate kinetics data showed a relatively low
, indicating a high affinity for substrate UDPGlc and also supported that 76E5 is more of a galactosyl and
-acetylglucosamine transferase. Sequence alignment and site-directed mutagenesis studies indeed suggested that serine is a crucial residue in the UDPGlcNAc and UDPGal activity.
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).
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