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Summary Sugar building blocks are crucial for the chemical diversity and biological activity of secondary metabolites. UDP‐dependent glycosyltransferases (UGTs) play a pivotal role in the biosynthesis of glycosides in plants by catalysing the attachment of sugar moieties to various bioactive natural products. However, the biosynthesis of oligosaccharide‐chain glycosides is often limited by the narrow substrate specificity of UGTs. In this study, we identify a regio‐specific β‐(1,6) glycosyltransferase, UGT94BY1, from Platycodon grandiflorum. UGT94BY1 exhibits broad substrate promiscuity and can transfer up to three sugar moieties to the C6‐OH position of the glucosyl group in various triterpenoids and phenolic glycosides, thereby forming β‐(1,6) oligoglucoside chains. To elucidate the mechanism underlying its substrate selectivity, we determined the crystal structure of the UGT94BY1 complex with UDP at a resolution of 2.0 Å. Molecular simulations revealed that a critical structural motif, comprising residues N84‐M91, S141‐L155 and R179‐E186, plays a key role in recognizing sugar acceptors and facilitating chain elongation. Our study unveils a powerful glycosyltransferase for β‐(1,6) oligoglucoside chain biosynthesis and highlights key regions involved in substrate recognition and sugar chain extension, providing valuable insights for designing UGTs with customized substrate specificities for biotechnological applications. We highlight a novel multi‐step O‐glycosyltransferase, UGT94BY1, which exhibits high promiscuity toward triterpene saponins and flavonoid glycosides for the formation of β‐(1,6) oligoglucoside chains. Through crystal structure analysis and theoretical calculations, we uncovered the mechanisms underlying substrate promiscuity and sugar chain extension.

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis,we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

The first sentence of the Funding Statement should read: \"This research was supported by grants from the National Natural Science Foundation of China (No. 91217301 to B.K.H.).\" (2013) Correction: UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

Plants possess an outer cell layer called the cell wall. This matrix comprises various molecules, such as polysaccharides and proteins, and serves a wide array of physiologically important functions. This structure is not static but rather flexible in response to the environment. One of the factors responsible for this plasticity is the xyloglucan endotransglucosylase/hydrolase (XTH) family, which cleaves and reconnects xyloglucan molecules. Since xyloglucan molecules have been hypothesised to tether cellulose microfibrils forming the main load-bearing network in the primary cell wall, XTHs have been thought to play a central role in cell wall loosening for plant cell expansion. However, multiple lines of recent evidence have questioned this classic model. Nevertheless, reverse genetic analyses have proven the biological importance of XTHs; therefore, a major challenge at present is to reconsider the role of XTHs inplanta. Recent advances in analytical techniques have allowed for gathering rich information on the structure of the primary cell wall. Thus, the integration of accumulated knowledge in current XTH studies may offer a turning point for unveiling the precise functions of XTHs. In the present review, we redefine the biological function of the XTH family based on the recent architectural model of the cell wall. We highlight three key findings regarding this enzyme family: (1) XTHs are not strictly required for cell wall loosening during plant cell expansion but play vital roles in response to specific biotic or abiotic stresses; (2) in addition to their transglycosylase activity, the hydrolase activity of XTHs is involved in physiological benefits; and (3) XTHs can recognise a wide range of polysaccharides other than xyloglucans.

• 2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins are one of the major saponin groups that are widely distributed in legumes such as pea, barrel medic, chickpea, and soybean. The steps involved in DDMP saponin biosynthesis remain uncharacterized at the molecular level. • We isolated two recessive mutants that lack DDMP saponins from an ethyl methanesulfonate-induced mutant population of soybean cultivar Pungsannamul. • Segregation analysis showed that the production of DDMP saponins is controlled by a single locus, named Sg-9. The locus was physically mapped to a 130-kb region on chromosome 16. Nucleotide sequence analysis of candidate genes in the region revealed that each mutant has a single-nucleotide polymorphism in the Glyma.16G033700 encoding a UDP-glycosyltransferase UGT73B4. Enzyme assays and mass spectrum-coupled chromatographic analysis reveal that the Sg-9 protein has glycosyltransferase activity, converting sapogenins and group B saponins to glycosylated products, and that mutant proteins had only partial activities. The tissue-specific expression profile of Sg-9 matches the accumulation pattern of DDMP saponins. • This is the first report on a new gene and its function in the biosynthesis of DDMP saponins. Our findings indicate that Sg-9 encodes a putative DDMP transferase that plays a critical role in the biosynthesis of DDMP saponins.

Background Dendrobium catenatum is a perennial herb of the genus Dendrobium orchidaceae . It has been known as “Golden Grass, Soft Gold” since ancient times with effects of strengthening the body, benefiting the stomach, generating body fluid, nourishing Yin and clearing internal heat. The flowers of D. catenatum have anti-oxidation, immune regulation and other biological activities. The composition analysis of flowers showed that flavonoid glycosides were significantly accumulated in floral tissue. However, in the flowers of D. catenatum , there was only one case of the UDP-glycosyltransferase (UGT) responsible for the glycosylation of flavonoids has been reported. Result In this study, a new UGT (named UGT708S6 ) was cloned from D. catenatum flowers rich in O -glycosides and C -glycosides, and its function and biochemical properties were characterized. Through homology comparison and molecular docking, we identified the key amino acid residues affecting the catalytic function of UGT708S6. The glycosyltransferase UGT708S6 was characterized and demonstrated C -glycosyltransferase (CGT) activity in vitro assay using phloretin and 2-hydroxynaringenin as sugar acceptors. The catalytic promiscuity assay revealed that UGT708S6 has a clear sugar donor preference, and displayed O -glycosyltransferase (OGT) activity towards luteolin, naringenin and liquiritigenin. Furthermore, the catalytic characteristics of UGT708S6 were explored, shedding light on the structural basis of substrate promiscuity and the catalytic mechanism involved in the formation of flavonoid C -glycosides. R271 was a key amino acid residue site that sustained the catalytic reaction. The smaller binding pocket resulted in the production of new O -glycosides and the reduction of C -glycosides. This highlighted the importance of the binding pocket in determining whether C -glycosides or O -glycosides were produced. Conclusions The findings suggest that UGT708S6 holds promise as a new glycosyltransferase for synthesizing flavonoid glycosides and offer valuable insights for further understanding the catalytic mechanisms of flavonoid glycosyltransferases.

Mogrosides constitute a series of natural sweeteners extracted from Siraitia grosvenorii fruits. These mogrosides are glucosylated to different degrees, with mogroside V (M5) and siamenoside I (SIA) being two mogrosides with high intensities of sweetness. SgUGT94-289-3 constitutes a uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) responsible for the biosynthesis of M5 and SIA, by continuously catalyzing glucosylation on mogroside IIe (M2E) and on the subsequent intermediate mogroside products. However, the mechanism of its promiscuous substrate recognition and multiple catalytic modes remains unclear. Here, we report multiple complex structures and the enzymatic characterization of the glycosyltransferase SgUGT94-289-3. We show that SgUGT94-289-3 adopts a dual-pocket organization in its active site, which allows the two structurally distinct reactive ends of mogrosides to be presented from different pockets to the active site for glucosylation reaction, thus enabling both substrate promiscuity and catalytic regioselectivity. We further identified a structural motif that is essential to catalytic activity and regioselectivity, and generated SgUGT94-289-3 mutants with greatly improved M5/SIA production from M2E in an in vitro one-pot setup. Here, the authors present structural and mechanistic characterisation of the UDP-dependent glycosyltransferase SgUGT94-289-3, which generates two mogrosides of interest as natural sweeteners. Structure-based engineering yielded variants with improved specificity and activity.

Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana . These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1–2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa , which is efficient at catalyzing β (1–2) glucosylation. The enzyme’s ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1–2) glucosylation in two distinct orientations as well as β (1–6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1–2) glucosylation, eliminate β (1–6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides. Steviol glycosides from the plant Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to facilitate the large-scale production of naturally rare but palatable glycosides Reb D and Reb M

Background Fusarium head blight (FHB), a devastating disease in wheat worldwide, results in yield loses and mycotoxin, such as deoxynivalenol (DON), accumulation in infected grains. DON also facilitates the pathogen colonization and spread of FHB symptoms during disease development. UDP-glycosyltransferase enzymes (UGTs) are known to contribute to detoxification and enhance FHB resistance by glycosylating DON into DON-3-glucoside (D3G) in wheat. However, a comprehensive investigation of wheat ( Triticum aestivum ) UGT genes is still lacking. Results In this study, we carried out a genome-wide analysis of family-1 UDP glycosyltransferases in wheat based on the PSPG conserved box that resulted in the identification of 179 putative UGT genes. The identified genes were clustered into 16 major phylogenetic groups with a lack of phylogenetic group K. The UGT genes were invariably distributed among all the chromosomes of the 3 genomes. At least 10 intron insertion events were found in the UGT sequences, where intron 4 was observed as the most conserved intron. The expression analysis of the wheat UGT genes using both online microarray data and quantitative real-time PCR verification suggested the distinct role of UGT genes in different tissues and developmental stages. The expression of many UGT genes was up-regulated after Fusarium graminearum inoculation, and six of the genes were further verified by RT-qPCR. Conclusion We identified 179 UGT genes from wheat using the available sequenced wheat genome. This study provides useful insight into the phylogenetic structure, distribution, and expression patterns of family-1 UDP glycosyltransferases in wheat. The results also offer a foundation for future work aimed at elucidating the molecular mechanisms underlying the resistance to FHB and DON accumulation.