Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation.

Sulcotrione is a member of triketone herbicides, a class of HPPD (4-hydroxyphenylpyruvate dioxygenase) inhibitors with broad-spectrum herbicidal activity. Modifications of glycosylation mediated by glycosyltransferases (GT) are involved in plant detoxification. In this study, we analyzed chip data published online and found that eight glycosyltransferases from group A of the apple glycosyltransferase family 1 may be involved in the metabolic mechanism of detoxification of triketone herbicides. To verify this prediction, we induced apple seedlings with six types of triketone herbicides, and then detected the expression levels of eight glycosyltransferase genes through real-time PCR. We found that triketone herbicides induced up-regulation of eight glycosyltransferase genes to varying degrees, with MdUGT91AJ2 being the most significantly up-regulated by sulcotrione-induced glycosyltransferase gene expression. Then, through in vitro enzymatic reactions and HPLC identification of glycoside substrates, it was found that the glycosyltransferase MdUGT91AJ2 had the highest specific enzyme activity against the triketone herbicide sulcotrione. Furthermore, the in vivo mechanism of the glycosyltransferase MdUGT91AJ2 in the detoxification metabolism of sulcotrione was further validated by overexpressing the strain in the plant. HPLC analysis showed that the content of sulcotrione glycosides in the overexpressing strain of MdUGT91AJ2 was significantly higher than that in the wild type. This result indicated that the apple glycosyltransferase MdUGT91AJ2 can still glycosylate and modify sulfotrione in plants, and participate in its detoxification metabolism. In summary, this study identified for the first time a novel apple glycosyltransferase MdUGT91AJ2 and elucidated its mechanism of action in the detoxification and metabolism of the triketone herbicide sulfotriene.

Aloesone is a bioactive natural product and biosynthetic precursor of rare glucosides found in rhubarb and some aloe plants including Aloe vera. This study aimed to investigate biocatalytic aloesone glycosylation and more than 400 uridine diphosphate-dependent glycosyltransferase (UGT) candidates, including multifunctional and promiscuous enzymes from a variety of plant species were assayed. As a result, 137 selective aloesone UGTs were discovered, including four from the natural producer rhubarb. Rhubarb UGT72B49 was further studied and its catalytic constants (kcat = 0.00092 ± 0.00003 s-1, KM = 30 ± 2.5 μM) as well as temperature and pH optima (50 °C and pH 7, respectively) were determined. We further aimed to find an efficient aloesone glycosylating enzyme with potential application for biocatalytic production of the glucoside. We discovered UGT71C1 from Arabidopsis thaliana as an efficient aloesone UGT showing a 167-fold higher catalytic efficiency compared to that of UGT72B49. Interestingly, sequence analysis of all the 137 newly identified aloesone UGTs showed that they belong to different phylogenetic groups, with the highest representation in groups B, D, E, F and L. Finally, our study indicates that aloesone C-glycosylation is highly specific and rare, since it was not possible to achieve in an efficient manner with any of the 422 UGTs assayed, including multifunctional GTs and 28 known C-UGTs.

Selenoneine, an ergothioneine analog, is important for antioxidation and detoxification. SenB and SenA are two crucial enzymes that form carbon-selenium bonds in the selenoneine biosynthetic pathway. To investigate their underlying catalytic mechanisms, we obtained complex structures of SenB with its substrate UDP-N-acetylglucosamine (UDP-GlcNAc) and SenA with N-α-trimethyl histidine (TMH). SenB adopts a type-B glycosyltransferase fold. Structural and functional analysis of the interaction network at the active center provide key information on substrate recognition and suggest a metal-ion-independent, inverting mechanism is utilized for SenB-mediated selenoglycoside formation. Moreover, the complex structure of SenA with TMH and enzymatic activity assays highlight vital residues that control substrate binding and specificity. Based on the conserved structure and substrate-binding pocket of the type I sulfoxide synthase EgtB in the ergothioneine biosynthetic pathway, a similar reaction mechanism was proposed for the formation of C-Se bonds by SenA. The structures provide knowledge on selenoneine synthesis and lay groundwork for further applications of this pathway.

Glycosylation plays a pivotal role in tuning the folding and function of proteins. Because most human therapeutic proteins are glycosylated, understanding and controlling glycosylation is important for the design, optimization, and manufacture of biopharmaceuticals. Unfortunately, natural eukaryotic glycosylation pathways are complex and often produce heterogeneous glycan patterns, making the production of glycoproteins with chemically precise and homogeneous glycan structures difficult. To overcome these limitations, bacterial glycoengineering has emerged as a simple, cost-effective, and scalable approach to produce designer glycoprotein therapeutics and vaccines in which the glycan structures are engineered to reduce heterogeneity and improve biological and biophysical attributes of the protein. Here, we discuss recent advances in bacterial cell-based and cell-free glycoengineering that have enabled the production of biopharmaceutical glycoproteins with customized glycan structures.

The Golgi apparatus, a crucial organelle involved in protein processing, including glycosylation, exhibits complex sub-structures, i.e., cis-, medial, and trans-cisternae. This study investigated the distribution of glycosyltransferases within the Golgi apparatus of mammalian cells via 3D super-resolution imaging. Focusing on human glycosyltransferases involved in N-glycan modification, we found that even enzymes presumed to coexist in the same Golgi compartment exhibit nuanced variations in localization. By artificially making their N-terminal regions [composed of a cytoplasmic, transmembrane, and stem segment (CTS)] identical, it was possible to enhance the degree of their colocalization, suggesting the decisive role of this region in determining the sub-Golgi localization of enzymes. Ultimately, this study reveals the molecular codes within CTS regions as key determinants of glycosyltransferase localization, providing insights into precise control over the positioning of glycosyltransferases, and consequently, the interactions between glycosyltransferases and substrate glycoproteins as cargoes in the secretory pathway. This study advances our understanding of Golgi organization and opens avenues for programming the glycosylation of proteins for clinical applications.Key words: Golgi apparatus, glycosyltransferase, 3D super-resolution imaging, N-glycosylation.

Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.

BACKGROUND: N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer\'s disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells.
METHODS: Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity.
RESULTS: Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases.
CONCLUSIONS: The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity.
CONCLUSIONS: Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.

Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes\' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.