Unravelling the relationship between substrate selectivity and primary sequence of UDP-glycosyltransferases

Plant natural products (NPs) are widely utilized in biotechnology, for example as fragrances, aromas, dyes and medicine. Although nature provides thousands of different NPs, only a small fraction of them is currently used in applications, partly because of problems in solubility and stability. These properties can be enhanced through glycosylation, but synthesis of glycosylated natural products is challenging. Enzymatic route to NP glycosylation is therefore of high interest. In plants, the enzymes responsible for NP glycosylation are called UDP-glycosyltransferases (UGTs) since they use UDP activated sugars as sugar donors. A single plant can have hundreds of UGTs allowing glycosylation of different compound groups. Understanding the bases of substrate selectivity would be important in allowing efficient engineering of UGTs for specific substrates and/or higher catalytic activities. Although UGTs have conserved tertiary structures, the relationship between UGT primary sequence and acceptor substrate is not well understood making enzyme engineering challenging. Main obstacles in creating a predictive model for substrate selectivity is the lag of UGT structures (currently nine plant UGT structures are available through PDB) and the lag of comparable information of UGT selectivity. Interestingly, it has been shown that UGT substrate selectivity is not related to phylogeny. Therefore, we wondered if more insights would be gained from comparing different phylogenetic groups to each other rather than trying to create a common predictive model for the whole enzyme group. By comparing structural information and sequence alignments, we indeed observed differences in substrate binding pocket folding when comparing UGTs from different phylogenetic groups. We hypothesize that this variation has led to difficulties in predicting substrate selectivity from UGT primary sequence, since some residues lining the binding pocket vary from one phylogenetic group to another. Therefore, it might be more feasible to predict substrate selectivity for each UGT phylogenetic group independently instead of the whole enzyme family

Biocatalytic synthesis of indigo and indican for blue denim dyeing

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X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion

Machine-learning based prediction of glycosyltransferase substrates

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Identification and functional characterization of novel plant UDP-glycosyltransferase (LbUGT72B10) for the bioremediation of 3,4-dichloroaniline

Herbicides are chemicals used to manipulate or control the growth of undesired plants and thereby protect crops. However, they and their degradation products can persist and accumulate in the environment, leading to contamination of soil and water systems and biodiversity loss. Interestingly, through the action of UDP-glycosyltransferases (UGTs), higher plants can glycosylate these xenobiotics, increasing their solubility and alleviating their toxicity. Here, seven plant UGTs belonging to family 72 of the UGT nomenclature were identified to N-glycosylate 3,4-dichloroaniline (3,4-DCA), which is a degradation product of commercially significant herbicides like Diuron, Linuron and Propanil. Although chlorinated chemicals are well-known UGT substrates, only one UGT with activity on 3,4-DCA (AtUGT72B1 from Arabidopsis thaliana) has been fully biochemically characterized. In this study, biochemical analysis revealed that six of the seven identified UGTs are capable of full conversion of 3,4-DCA to its N-glucoside. The most efficient enzyme was found to be LbUGT72B10 from Lycium barbarum (kcat = 11.2 s−1, KM = 51.2 μM). Consequently, transgenic expression of LbUGT72B10 could potentially play a role in the future in the mitigation of 3,4-DCA toxicity, preventing its accumulation in living systems and reducing contamination of waterways and soil

Functional characterization of the phosphotransferase system in <i>Parageobacillus thermoglucosidasius</i>

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis

The function of UDP-glycosyltransferases in plants and their possible use in crop protection

Glycosyltransferases catalyse the transfer of a glycosyl moiety from a donor to an acceptor. Members of this enzyme class are ubiquitous throughout all kingdoms of life and are involved in the biosynthesis of countless types of glycosides. Family 1 glycosyltransferases, also referred to as uridine diphosphate-dependent glycosyltransferases (UGTs), glycosylate small molecules such as secondary metabolites and xenobiotics. In plants, UGTs are recognised for their multiple functionalities ranging from roles in growth regulation and development, in protection against pathogens and abiotic stresses and in adaptation to changing environments. In this study, we review UGT-mediated glycosylation of phytohormones, endogenous secondary metabolites, and xenobiotics and contextualise the role this chemical modification plays in the response to biotic and abiotic stresses and plant fitness. Here, the potential advantages and drawbacks of altering the expression patterns of specific UGTs along with the heterologous expression of UGTs across plant species to improve stress tolerance in plants are discussed. We conclude that UGT-based genetic modification of plants could potentially enhance agricultural efficiency and take part in controlling the biological activity of xenobiotics in bioremediation strategies. However, more knowledge of the intricate interplay between UGTs in plants is needed to unlock the full potential of UGTs in crop resistance.</p