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

The sugar donor specificity of plant family 1 glycosyltransferases

Plant 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

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