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Identification and functional characterization of novel plant UDP-glycosyltransferase (LbUGT72B10) for the bioremediation of 3,4-dichloroaniline
No full textHerbicides 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
Thermostable glycosyltransferase variants
No full textThe present invention concerns glycosyltransferase enzyme mutants having improved half-lives and thermal stability compared to the parent enzyme UDP-glycosyltransferase (PtUGT) from the indigo producing plant Polygonum tinctorium/Persicaria tinctoria; and further provides a composition, kit, and methods employing these mutants for glycosylation of desired compounds, such as indoxyl compounds.</p
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Molecular characteristics of plant UDP-arabinopyranose mutases
No full textl-arabinofuranose is a ubiquitous component of the cell wall and various natural products in plants, where it is synthesized from cytosolic UDP-arabinopyranose (UDP-Arap). The biosynthetic machinery long remained enigmatic in terms of responsible enzymes and subcellular localization. With the discovery of UDP-Arap mutase in plant cytosol, the demonstration of its role in cell-wall arabinose incorporation and the identification of UDP-arabinofuranose transporters in the Golgi membrane, it is clear that the cytosolic UDP-Arap mutases are the key enzymes converting UDP-Arap to UDP-arabinofuranose for cell wall and natural product biosynthesis. This has recently been confirmed by several genotype/phenotype studies. In contrast to the solid evidence pertaining to UDP-Arap mutase function in vivo, the molecular features, including enzymatic mechanism and oligomeric state, remain unknown. However, these enzymes belong to the small family of proteins originally identified as reversibly glycosylated polypeptides (RGPs), which has been studied for >20 years. Here, we review the UDP-Arap mutase and RGP literature together, to summarize and systemize reported molecular characteristics and relations to other proteins
The sugar donor specificity of plant family 1 glycosyltransferases
Get PDFPlant family 1 glycosyltransferases (UGTs) represent a formidable tool to produce valuable natural and novel glycosides. Their regio- and stereo-specific one-step glycosylation mechanism along with their inherent wide acceptor scope are desirable traits in biotechnology. However, their donor scope and specificity are not well understood. Since different sugars have different properties in vivo and in vitro, the ability to easily glycodiversify target acceptors is desired, and this depends on our improved understanding of the donor binding site. In the aim to unlock the full potential of UGTs, studies have attempted to elucidate the structure-function relationship governing their donor specificity. These efforts have revealed a complex phenomenon, and general principles valid for multiple enzymes are elusive. Here, we review the studies of UGT donor specificity, and attempt to group the information into key concepts which can help shape future research. We zoom in on the family-defining PSPG motif, on two loop residues reported to interact with the C6 position of the sugar, and on the role of active site arginines in donor specificity. We continue to discuss attempts to alter and expand the donor specificity by enzyme engineering, and finally discuss future research directions
The function of UDP-glycosyltransferases in plants and their possible use in crop protection
No full textGlycosyltransferases 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
Natural product C-glycosyltransferases - a scarcely characterised enzymatic activity with biotechnological potential
No full textCovering: up to 2020C-Glycosyltransferases are enzymes that catalyse the transfer of sugar molecules to carbon atoms in substituted aromatic rings of a variety of natural products. The resulting β-C-glycosidic bond is more stable in vivo than most O-glycosidic bonds, hence offering an attractive modulation of a variety of compounds with multiple biological activities. While C-glycosylated natural products have been known for centuries, our knowledge of corresponding C-glycosyltransferases is scarce. Here, we discuss commonalities and differences in the known C-glycosyltransferases, review attempts to leverage them as synthetic biocatalysts, and discuss current challenges and limitations in their research and application
Family 1 glycosyltransferases (GT1, UGTs) are subject to dilution-induced inactivation and low chemo stability toward their own acceptor substrates
No full textGlycosylation reactions are essential but challenging from a conventional chemistry standpoint. Conversely, they are biotechnologically feasible as glycosyltransferases can transfer sugar to an acceptor with perfect regio- and stereo-selectivity, quantitative yields, in a single reaction and under mild conditions. Low stability is often alleged to be a limitation to the biotechnological application of glycosyltransferases. Here we show that these enzymes are not necessarily intrinsically unstable, but that they present both dilution-induced inactivation and low chemostability towards their own acceptor substrates, and that these two phenomena are synergistic. We assessed 18 distinct GT1 enzymes against three unrelated acceptors (apigenin, resveratrol, and scopoletin—respectively a flavone, a stilbene, and a coumarin), resulting in a total of 54 enzymes: substrate pairs. For each pair, we varied catalyst and acceptor concentrations to obtain 16 different reaction conditions. Fifteen of the assayed enzymes (83%) displayed both low chemostability against at least one of the assayed acceptors at submillimolar concentrations, and dilution-induced inactivation. Furthermore, sensitivity to reaction conditions seems to be related to the thermal stability of the enzymes, the three unaffected enzymes having melting temperatures above 55°C, whereas the full enzyme panel ranged from 37.4 to 61.7°C. These results are important for GT1 understanding and engineering, as well as for discovery efforts and biotechnological use
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