Glycosyltransferase Research Articles

A Genome-Centric Approach Reveals a Novel Glycosyltransferase from the GA A07 Strain of Bacillus thuringiensis Responsible for Catalyzing 15-O-Glycosylation of Ganoderic Acid A.

Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-β-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-β-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.

Investigation of the conserved reentrant membrane helix in the monotopic phosphoglycosyl transferase superfamily supports key molecular interactions with polyprenol phosphate substrates

Long-chain polyprenol phosphates feature in membrane-associated glycoconjugate biosynthesis pathways across domains of life. These unique amphiphilic molecules are best known as substrates of polytopic membrane proteins, including polyprenol-phosphate phosphoglycosyl and glycosyl transferases, and as components of more complex substrates. The linear polyprenols are constrained by double bond geometry and lend themselves well to interactions with polytopic membrane proteins, in which multiple transmembrane helices form a rich landscape for interactions. Recently, a new superfamily of monotopic phosphoglycosyl transferase enzymes has been identified that interacts with polyprenol phosphate substrates via a single reentrant membrane helix. Intriguingly, despite the dramatic differences in their membrane-interaction domains, both polytopic and monotopic enzymes similarly favor a unique cis/trans geometry in their polyprenol phosphate substrates.Herein, we present a multipronged biochemical and biophysical study of PglC, a monotopic phosphoglycosyl transferase that catalyzes the first membrane-committed step in N-linked glycoprotein biosynthesis in Campylobacter jejuni. We probe the significance of polyprenol phosphate geometry both in mediating substrate binding to PglC and in modulating the local membrane environment. Geometry is found to be important for binding to PglC; a conserved proline residue in the reentrant membrane helix is determined to drive polyprenol phosphate recognition and specificity. Pyrene fluorescence studies show that polyprenol phosphates at physiologically-relevant levels increase the disorder of the local lipid bilayer; however, this effect is confined to polyprenol phosphates with specific isoprene geometries. The molecular insights from this study may shed new light on the interactions of polyprenol phosphates with diverse membrane-associated proteins in glycoconjugate biosynthesis.

A New Triterpenoid Glucoside from a Novel Acidic Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis.

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-β-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-β-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-β-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min−1) was 3-fold higher than that at pH 7 (kcat = 3.8 min−1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.

Genome-Wide Identification and Characterization of Glycosyltransferase Family 47 in Cotton

The glycosyltransferase (GT) 47 family is involved in the biosynthesis of xylose, pectin and xyloglucan and plays a significant role in maintaining the normal morphology of the plant cell wall. However, the functions of GT47s are less well known in cotton. In the present study, a total of 53, 53, 105 and 109 GT47 genes were detected by genome-wide identification in Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. All the GT47s were classified into six major groups via phylogenetic analysis. The exon/intron structure and protein motifs indicated that each branch of the GT47 genes was highly conserved. Collinearity analysis showed that GT47 gene family expansion occurred in Gossypium spp. mainly through whole-genome duplication and that segmental duplication mainly promoted GT47 gene expansion within the A and D subgenomes. The Ka/Ks values suggested that the GT47 gene family has undergone purifying selection during the long-term evolutionary process. Transcriptomic data and qRT-PCR showed that GhGT47 genes exhibited different expression patterns in each tissue and during fiber development. Our results suggest that some genes in the GhGT47 family might be associated with fiber development and the abiotic stress response, which could promote further research involving functional analysis of GT47 genes in cotton.

Molecular Basis of ABO Variants Including Identification of 16 Novel ABO Subgroup Alleles in Chinese Han Population

Introduction: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion. Methods: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software. Results: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO*A alleles, 23 ABO*B alleles, 4 ABO*BA alleles, 4 ABO*cisAB alleles, and 1 ABO*O alleles. ABO*A2.08 and ABO*BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression. Conclusions: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety.

Research progress in understanding the structure, mechanism, and engineering of plant glycosyltransferases

Glycosyltransferases (GTs) are found in a wide variety of organisms, where they are involved in maintaining cell metabolism through glycosylation reactions. Glycosyltransferases transfer activated sugar from donor molecules to a variety of acceptors, this can alter the hydrophilic properties, stability, and chemical properties as well as subcellular location and biological activity of the acceptors. Many compounds that are modified by glycosylation are important sources of drug molecules. However, natural products contain only small amounts of glycosylated compounds, and it is very difficult to extract and purify these compounds. Furthermore, during the chemical synthesis of glycosylated compounds, the raw material reagents and generated by-products tend to cause environmental pollution, and it is not possible to achieve glycosylation at the particular acceptor sites. Recently, GTs have garnered interest among researchers. Therefore, here we review the current structural studies and biotechnological applications of plant GTs that would provide useful information for glycosylation engineering and bioactive glycoside production.

EXT1, Regulated by MiR-665, Promotes Cell Apoptosis via ERK1/2 Signaling Pathway in Acute Lymphoblastic Leukemia

BackgroundEXT1 is an endoplasmic reticulum-resident glycosyl transferase whose intracellular expression alters the biosynthesis and distribution of heparan sulfate. EXT1 is regarded as a classic tumor suppressor. MiR-665 can act as either an oncogene or tumor-suppressing gene in different tumors. The aim of the current study was to determine the function and molecular mechanisms of EXT1 and miR-665 in acute lymphoblastic leukemia (ALL).Material/MethodsEXT1 expression in ALL was evaluated by real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of EXT1 in ALL were explored by Cell Counting Kit-8 (CCK-8)/EdU assays, western blotting, flow cytometry, and in vivo tumorigenesis assays. Label-free quantification was used to detect differentially expressed proteins in EXT1-overexpressing Reh cells.ResultsEXT1 expression is downregulated in ALL and negatively correlated with miR-665 expression. Moreover, low EXT1 and high miR-665 expression levels in adult ALL bone marrow tissues are correlated with poor patient survival. Our study showed that EXT1 modulates the proliferation and apoptosis of ALL cells in vitro and in vivo and that miR-665 promotes cell growth and inhibits apoptosis by suppressing EXT1. EXT1 promotes cell apoptosis via deactivating the ERK1/2 pathway.ConclusionsIn conclusion, this study is the first to confirm the association between low EXT1 levels and several clinical features of ALL. Low bone marrow EXT1 levels independently predict poor prognoses in adult ALL patients. Thus, our study suggests that EXT1- or miR-665-targeted strategies can confer the therapeutic effect of promoting apoptosis by deactivating the ERK1/2 pathway.

High-Throughput Approaches in Carbohydrate-Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery.

Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

AbstractCarbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.

Critical Review of Plant Cell Wall Matrix Polysaccharide Glycosyltransferase Activities Verified by Heterologous Protein Expression.

The life cycle and development of plants requires the biosynthesis, deposition, and degradation of cell wall matrix polysaccharides. The structures of the diverse cell wall matrix polysaccharides influence commercially important properties of plant cells, including growth, biomass recalcitrance, organ abscission, and the shelf life of fruits. This review is a comprehensive summary of the matrix polysaccharide glycosyltransferase (GT) activities that have been verified using in vitro assays following heterologous GT protein expression. Plant cell wall (PCW) biosynthetic GTs are primarily integral transmembrane proteins localized to the endoplasmic reticulum and Golgi of the plant secretory system. The low abundance of these enzymes in plant tissues makes them particularly difficult to purify from native plant membranes in quantities sufficient for enzymatic characterization, which is essential to study the functions of the different GTs. Numerous activities in the synthesis of the major cell wall matrix glycans, including pectins, xylans, xyloglucan, mannans, mixed-linkage glucans (MLGs), and arabinogalactan components of AGP proteoglycans have been mapped to specific genes and multi-gene families. Cell wall GTs include those that synthesize the polymer backbones, those that elongate side branches with extended glycosyl chains, and those that add single monosaccharide linkages onto polysaccharide backbones and/or side branches. Three main strategies have been used to identify genes encoding GTs that synthesize cell wall linkages: analysis of membrane fractions enriched for cell wall biosynthetic activities, mutational genetics approaches investigating cell wall compositional phenotypes, and omics-directed identification of putative GTs from sequenced plant genomes. Here we compare the heterologous expression systems used to produce, purify, and study the enzyme activities of PCW GTs, with an emphasis on the eukaryotic systems Nicotiana benthamiana, Pichia pastoris, and human embryonic kidney (HEK293) cells. We discuss the enzymatic properties of GTs including kinetic rates, the chain lengths of polysaccharide products, acceptor oligosaccharide preferences, elongation mechanisms for the synthesis of long-chain polymers, and the formation of GT complexes. Future directions in the study of matrix polysaccharide biosynthesis are proposed.

Life history and functional capacity of the microbiome are altered in beta-cypermethrin-resistant cockroaches

Cockroaches are widely perceived to evolve resistance to insecticides. Over-expression of a resistance-conferring gene can be costly and may require energy and resource reallocation for metabolic and developmental processes. To evaluate whether changes in the composition of gut microbiota in Blattella germanica affected its resistance evolution to beta-cypermethrin and to determine the role of gut microbiota in host growth and development, we studied the relationship between insecticide resistance and the diversity and genetic content of gut microbiota in cockroaches. Results suggest beta-cypermethrin-resistant cockroaches (R strain) exhibited a delayed development period and reduced adult longevity compared with susceptible cockroaches (S strain). Based on 16S rRNA gene sequencing and community metagenomics, we found that the relative abundance of Lactobacillus and Acetobacteraceae were significantly lower in the R strain compared with the S strain in the foregut and midgut of both strains. Functional annotation of Kyoto Encyclopedia of Genes and Genomes (KEGG) modules of midgut genes in the two strains revealed that 10.6% were involved in metabolism, while the relative abundance in the R strain was 7.4%. Unigenes were also translated into amino acid sequences and assigned to protein families based on hits to the Carbohydrate-Active enzymes (CAZy) database. This process identified the glycoside hydrolases, glycosyl transferases and carbohydrate-binding modules of the S strain as all being significantly higher in diversity than those in the R strain. Overall, we conclude that fitness-related costs increased in the resistant strain of cockroaches compared with the susceptible strain, and the variation in insect gut microbiota, especially those related to growth and development, was an important influencing factor.

Identification of carbohydrate active enzymes from whole genome sequence of Tilletia indica and sporulation analysis

Karnal bunt of wheat is an internationally quarantined disease. One of the major constraints in exporting wheat is prevalence of Karnal bunt (KB). It is caused by Tilletia indica which is a heterothallic fungus and requires fusion of different mating types for causing infection. The growth efficiency and aggressiveness of plant pathogens are often associated with their carbohydrate active enzymes (CAZymes). The present study was undertaken to identify the CAZymes in whole genome sequence of Tilletia indica, and conduct sporulation analysis. Total 315 predicted secretory proteins were annotated with the CAZyme database (dbCAN). The secreted carbohydrates active enzymes were identified as 105 glycosylhydrolases (GH), 85 glycosyltransferases (GT), 83 carbohydrate esterases (CE), 8 carbohydrate binding modules (CBM), 30 auxiliary activities (AA) and 4 polysaccharide lyases (PL). Based on sporulation analysis of T. indica isolates, KB1 and KB2 isolates were most sporulative among all the isolates of T. indica. These carbohydrate active enzymes can help to understand the pathogenesis mechanism(s) of Karnal bunt using functional genomics approach.

RQ3, A Natural Rebaudioside D Isomer, Was Obtained from Glucosylation of Rebaudioside A Catalyzed by the CGTase Toruzyme 3.0 L.

In this study, a monoglucosyl rebaudioside A product was isolated from the mixture of glucosylated rebaudioside A obtained from the most reported and industrial used cyclodextrin glycosyl transferase, Toruzyme 3.0 L (CGTase, Toruzyme 3.0 L). The molecular structure of the monoglucosyl rebaudioside A was characterized using LC-MS/MS and methylation analysis combined with 1D and 2D NMR, indicating that it is 13-[(2-O-(3-α-O-D-glucopyranosyl)-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (also known as RQ3, which naturally exists in Stevia extract as an isomer of rebaudioside D). This study may help to further understand the reaction mechanism of glucosylation of steviol glycoside assisted by Toruzyme 3.0 L in the aspect of molecule linkage pattern, and also benefit theapplication of the glucosylated rebaudiosides.

Transcriptomic Analysis Identifies New Non-Target Site Glyphosate-Resistance Genes in Conyza bonariensis.

Conyza bonariensis (hairy fleabane) is one of the most problematic and widespread glyphosate-resistant weeds in the world. This highly competitive weed species significantly interferes with crop growth and substantially decreases crop yield. Despite its agricultural importance, the molecular mechanisms of glyphosate resistance are still unknown. The present RNA-Seq study was performed with the goal of identifying differentially expressed candidate transcripts (genes) related to metabolism-based non-target site glyphosate resistance in C. bonariensis. The whole-transcriptome was de novo assembled from glyphosate-resistant and -sensitive biotypes of C. bonariensis from Southern Brazil. The RNA was extracted from untreated and glyphosate-treated plants at several timepoints up to 288 h after treatment in both biotypes. The transcriptome assembly produced 90,124 contigs with an average length of 777 bp and N50 of 1118 bp. In response to glyphosate treatment, differential gene expression analysis was performed on glyphosate-resistant and -sensitive biotypes. A total of 9622 genes were differentially expressed as a response to glyphosate treatment in both biotypes, 4297 (44.6%) being up- and 5325 (55.4%) down-regulated. The resistant biotype presented 1770 up- and 2333 down-regulated genes while the sensitive biotype had 2335 and 2800 up- and down-regulated genes, respectively. Among them, 974 up- and 1290 down-regulated genes were co-expressed in both biotypes. In the present work, we identified 41 new candidate target genes from five families related to herbicide transport and metabolism: 19 ABC transporters, 10 CYP450s, one glutathione S-transferase (GST), five glycosyltransferases (GT), and six genes related to antioxidant enzyme catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). The candidate genes may participate in metabolic-based glyphosate resistance via oxidation, conjugation, transport, and degradation, plus antioxidation. One or more of these genes might ‘rescue’ resistant plants from irreversible damage after glyphosate treatment. The 41 target genes we report in the present study may inform further functional genomics studies, including gene editing approaches to elucidate glyphosate-resistance mechanisms in C. bonariensis.

Mechanism of oxytetracycline removal by aerobic granular sludge in SBR

In this study, oxytetracycline (OTC) as a target pollutant in swine wastewater was removed by aerobic granular sludge (AGS). The removal rate of 300 μg/L OTC in aerobic granular sludge sequencing batch reactor (AGSBR) increased to 88.00% in 33 days and maintained stable. The chemical oxygen demand (COD), ammonium nitrogen (NH4+-N) and total phosphorus (TP) in wastewater were also efficiently removed. The removal of OTC mainly depended on the adsorption and biodegradation of AGS, and the biodegradation was increased obviously after AGS adaptation to OTC. The degradation products of OTC were analyzed by mass spectrometry. The analysis of metagenome sequencing revealed that the enzymes, such as glycosyl transferases (GTs), polysaccharide lyases (PLs) and auxiliary activities (AAs), may play an important role in the removal of OTC. The Lefse analysis showed that the Flavobacteriia, Flavobacteriales, Cryomorphaceae and Fluviicola were four kinds of microbes with significant difference in OTC feed reactor, which are considered to be drug-resistant bacteria in AGSBR. Furthermore, the dynamics of microbial community changed significantly at three levels, including the enrichment of drug-resistant microorganisms and the microorganisms that gradually reduced or even disappeared under the pressure of OTC.

Three Capsular Polysaccharide Synthesis-Related Glucosyltransferases, GT-1, GT-2 and WcaJ, Are Associated With Virulence and Phage Sensitivity of Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

Phylogeny-guided characterization of glycosyltransferases for epothilone glycosylation.

SummaryGlycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti‐tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC‐subbranch with 160 GT sequences. We demonstrated that the YjiC‐subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC‐subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high‐active GTs (Q66 and P77) but changed in two low‐efficient GTs. Site‐directed residues swapping at the two positions in the two low‐active GTs (BssGT and BamGT) and the high‐active GT BsGT‐1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

Identification of a UDP-Glucosyltransferase favouring substrate- and regio-specific biosynthesis of flavonoid glucosides in Cyclocarya paliurus

Cyclocarya paliurus (Batalin) Iljinsk is a medicinal plant belonging to the Juglandaceae family, and its leaves are used for a traditional sweet herbal tea with bioactivity against obesity and hyperglycaemia in China. It contains various bioactive specialised metabolites, such as flavonoids, triterpenes and their glucosides, while no glycosyltransferases (GTs) have been reported in C. paliurus to date. Herein, we identified and cloned the first glucosyltransferase C. paliurus GT1. The expression profiles of C. paliurus GT1 showed very high expression in young leaves, callus and branches, but relatively low expression in old leaves and bark and no expression in root. The recombinant C. paliurus GT1 protein was heterologously expressed in Escherichia coli and exhibited catalytic activity towards multiple flavonoids favouring substrate- and regio-specific biosynthesis. Further enzyme assays indicated a preference for certain hydroxyl group glucosylation by C. paliurus GT1. C. paliurus GT1 actively catalysed the glucosylation of flavones and flavonols, but it was less active towards isoflavones, flavanones or triterpenes. C. paliurus GT1 was also able to catalyse the attachment of sugars to the thiol (S-) or amine (N-) sites on aromatic compounds but not on aliphatic compounds. Molecular docking and site-directed mutagenesis analyses indicated that A43F, V84P, and M201Y dramatically altered the regio-selectivity and activity, and the W283M mutation and deletion of the V309-D320 region enhanced the activity and the formation of disaccharides. Herein, we present the identification and characterization of the first multi-functional glucosyltransferase in C. paliurus and provide a basis for understanding the biosynthesis of flavonoid glucosides. C. paliurus GT1 could be utilized as a synthetic biology tool for the synthesis of O-, N-, or S-glucosylated natural/unnatural products.

Structure and mechanism of TagA, a novel membrane-associated glycosyltransferase that produces wall teichoic acids in pathogenic bacteria.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme’s lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a β-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a β-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.