Novel missense mutation c.797T>C (p.Met266Thr) gives rise to the rare B(A) phenotype in a Chinese family.

BACKGROUND AND OBJECTIVES: B(A) phenotype is usually formed by nucleotide mutations in the ABO*B.01 allele, with their products exhibiting glycosyltransferases (GTs) A and B overlapping functionality. We herein report a B(A) allele found in a Chinese family. MATERIALS AND METHODS: The entire ABO genes of the probands, including flanking regulatory regions, were sequenced through PacBio third-generation long-read single-molecule real-time sequencing. 3D molecular models of the wild-type and mutant GTB were generated using the DynaMut web server. The effect of the mutation on the enzyme function was predicted by PROVEAN and PolyPhen2. The predictions of stability changes were performed using DynaMut and SNPeffect. RESULTS: Based on serological and sequencing features, we concluded the two probands as possible cases of the B(A) phenotype. Crystallization analysis showed that Thr266 substitution does not disrupt the hydrogen bonds. However, some changes in interatomic contacts, such as loss of ionic interactions and hydrophobic contacts, and addition of weak hydrogen bonds, may have affected protein stability to some extent. This mutation was predicted to have a benign effect on enzyme function and slightly reduce protein stability. CONCLUSION: The probands had the same novel B(A) allele with a c.797T>C (p.Met266Thr) mutation on the ABO*B.01 backbone.

The role of UDP-glycosyltransferases in xenobioticresistance.

Uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) are an enzyme superfamily that catalyzes glycosyl residues transfer from activated nucleotide sugars to acceptor molecules. In addition to various endogenous compounds, numerous xenobiotics are substrates of UGTs. As the glycosides formed are generally less active/toxic and more hydrophilic than aglycones, UGTs effectively protect organisms from potentially harmful xenobiotics. Therefore, increased UGT expression and/or activity improve the protection of the organism and may contribute to the development of individuals that become more resistant to certain xenobiotics. While the function of UGTs in the resistance of human cancer cells to chemotherapy is now well known, other organisms and other xenobiotics have attracted much less attention. This review was designed to fill this knowledge gap by presenting complex information about the role of UGTs in xenobiotic-resistance in various organisms. This summarization and evaluation of the available information reveals that UGTs play an important role in defense against xenobiotics not only in humans, but in countless other organisms such as parasites, insects, and plants. Moreover, many recent studies clearly show the participation of UGTs in the resistance of nematodes to anthelmintics, insects to insecticides, weeds to herbicides as well as humans to various drugs (not only those used in cancer therapy but also in the treatment of epilepsy, psychiatric disorders, hypertension, hypercholesterolemia, and HIV infection). Nevertheless, although the contribution of UGTs to xenobiotic resistance in diverse organisms has become obvious, many pieces of information remain missing, for example with regard to the mechanisms of UGT regulation.

Characterization of Three Paris polyphylla Glycosyltransferases from Different UGT Families for Steroid Functionalization.

Plant steroid glycosides, such as phytosterol glycosides, steroidal saponins, and steroidal glycoalkaloids, are natural products with great pharmaceutical values. In this study, we characterized three UDP-glycosyltransferases (UGTs) involved in the glycosylation of steroidal sapogenin from Paris polyphylla. Substrate specificity analysis revealed that UGT73CR1 could glycosylate steroidal sapogenins and steroidal alkaloids, with the highest affinity for diosgenin. The residues His27 and Asp129 of UGT73CR1 are conserved in corresponding positions of plant glycosyltransferases, which are crucial for activating the C-3 OH of the receptor substrates. In comparison, UGT80A33 and UGT80A34 exhibited a higher affinity for cholesterol than other steroids. UGT80s have a larger active pocket, which allows them to accommodate the side chain of sterols. In summary, we assessed three P. polyphylla glycosyltransferases from two UGT families for the functionalization of steroidal molecules, which will provide a basis for the future biomanufacturing of diverse bioactive steroid glycosides.

Structural basis for substrate recognition in the Phytolacca americana glycosyltransferase PaGT3.

Capsaicinoids are phenolic compounds that have health benefits. However, the pungency and poor water solubility of these compounds limit their exploitation. Glycosylation is a powerful method to improve water solubility and reduce pungency while preserving bioactivity. PaGT3, a uridine diphosphate glycosyltransferase (UGT) from Phytolacca americana, is known for its ability to glycosylate capsaicinoids and other phenolic compounds. While structural information on several UGTs is available, structures of UGTs that can glycosylate a range of phenolic compounds are rare. To fill this gap, crystal structures of PaGT3 with a sugar-donor analogue (UDP-2-fluoroglucose) and the acceptors capsaicin and kaempferol were determined. PaGT3 adopts a GT-B-fold structure that is highly conserved among UGTs. However, the acceptor-binding pocket in PaGT3 is hydrophobic and large, and is surrounded by longer loops. The larger acceptor-binding pocket in PaGT3 allows the enzyme to bind a range of compounds, while the flexibility of the longer loops possibly plays a role in accommodating the acceptors in the binding pocket according to their shape and size. This structural information provides insights into the acceptor-binding mechanism in UGTs that bind multiple substrates.

Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity.

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein WzySF polymerises Oag repeat units, whilst two co-polymerase proteins, WzzSF and WzzpHS-2, together interact with WzySF to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of WzySF transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of WzySF, or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ WzySF arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe WzySF conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including WzzSF and WzzpHS-2, and extrinsic factors, such as temperature. Site-directed mutagenesis of WzySF cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent analyses highlighted both the structural and functional importance of WzySF cysteines. Substitution of WzySF cysteine residues significantly decreased biosynthesis of the VL-Oag modal length, without disruption to S-Oag production. This phenotype was corroborated in the absence of co-polymerase competition for WzySF interaction. These data suggest WzySF cysteine substitutions directly impair the interaction between Wzy/WzzpHS-2, without altering the Wzy/WzzSF interplay, and in combination with structural data, we propose that the N- and C-termini of WzySF are arranged in close proximity, and together may form the unique WzzpHS-2 interaction site.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA.

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and ß-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural C-glycoside nothofagin.

C-glycosyltransferase (CGT) and sucrose synthase (SuSy), each fused to the cationic binding module Zbasic2 , were co-immobilized on anionic carrier (ReliSorb SP400) and assessed for continuous production of the natural C-glycoside nothofagin. The overall reaction was 3'-C-ß-glycosylation of the polyphenol phloretin from uridine 5'-diphosphate (UDP)-glucose that was released in situ from sucrose and UDP. Using solid catalyst optimized for total (∼28 mg/g) as well as relative protein loading (CGT/SuSy = ∼1) and assembled into a packed bed (1 ml), we demonstrate flow synthesis of nothofagin (up to 52 mg/ml; 120 mM) from phloretin (≥95% conversion) solubilized by inclusion complexation in hydroxypropyl ß-cyclodextrin. About 1.8 g nothofagin (90 ml; 12-26 mg/ml) were produced continuously over 90 reactor cycles (2.3 h/cycle) with a space-time yield of approximately 11 mg/(ml h) and a total enzyme turnover number of up to 2.9 × 103 mg/mg (=3.8 × 105 mol/mol). The co-immobilized enzymes exhibited useful effectiveness (∼40% of the enzymes in solution), with limitations on the conversion rate arising partly from external liquid-solid mass transfer of UDP under packed-bed flow conditions. The operational half-life of the catalyst (∼200 h; 30°C) was governed by the binding stability of the glycosyltransferases (≤35% loss of activity) on the solid carrier. Collectively, the current study shows integrated process technology for flow synthesis with co-immobilized sugar nucleotide-dependent glycosyltransferases, using efficient glycosylation from sucrose via the internally recycled UDP-glucose. This provides a basis from engineering science to promote glycosyltransferase applications for natural product glycosides and oligosaccharides.

Structural and mechanistic investigations of protein S-glycosyltransferases.

Attachment of sugars to nitrogen and oxygen in peptides is ubiquitous in biology, but glycosylation of sulfur atoms has only been recently described. Here, we characterize two S-glycosyltransferases SunS and ThuS that selectively glycosylate one of five Cys residues in their substrate peptides; substitution of this Cys with Ser results in a strong decrease in glycosylation activity. Crystal structures of SunS and ThuS in complex with UDP-glucose or a derivative reveal an unusual architecture in which a glycosyltransferase type A (GTA) fold is decorated with additional domains to support homodimerization. Dimer formation creates an extended cavity for the substrate peptide, drawing functional analogy with O-glycosyltransferases involved in cell wall biosynthesis. This extended cavity contains a sharp bend that may explain the site selectivity of the glycosylation because the target Cys is in a Gly-rich stretch that can accommodate the bend. These studies establish a molecular framework for understanding the unusual S-glycosyltransferases.

The surprising structural and mechanistic dichotomy of membrane-associated phosphoglycosyl transferases.

Phosphoglycosyl transferases (PGTs) play a pivotal role at the inception of complex glycoconjugate biosynthesis pathways across all domains of life. PGTs promote the first membrane-committed step in the en bloc biosynthetic strategy by catalyzing the transfer of a phospho-sugar from a nucleoside diphospho-sugar to a membrane-resident polyprenol phosphate. Studies on the PGTs have been hampered because they are integral membrane proteins, and often prove to be recalcitrant to expression, purification and analysis. However, in recent years exciting new information has been derived on the structures and the mechanisms of PGTs, revealing the existence of two unique superfamilies of PGT enzymes that enact catalysis at the membrane interface. Genome neighborhood analysis shows that these superfamilies, the polytopic PGT (polyPGT) and monotopic PGT (monoPGT), may initiate different pathways within the same organism. Moreover, the same fundamental two-substrate reaction is enacted through two different chemical mechanisms with distinct modes of catalysis. This review highlights the structural and mechanistic divergence between the PGT enzyme superfamilies and how this is reflected in differences in regulation in their varied glycoconjugate biosynthesis pathways.

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases.

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Crown-ether-mediated crystal structures of the glycosyltransferase PaGT3 from Phytolacca americana.

Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structure-function relationship of PaGT3, its crystal structure was determined. The sugar-donor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptor-binding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein. The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.

How glycosylation affects glycosylation: the role of N-glycans in glycosyltransferase activity.

N-glycosylation is one of the most important posttranslational modifications of proteins. It plays important roles in the biogenesis and functions of proteins by influencing their folding, intracellular localization, stability and solubility. N-glycans are synthesized by glycosyltransferases, a complex group of ubiquitous enzymes that occur in most kingdoms of life. A growing body of evidence shows that N-glycans may influence processing and functions of glycosyltransferases, including their secretion, stability and substrate/acceptor affinity. Changes in these properties may have a profound impact on glycosyltransferase activity. Indeed, some glycosyltransferases have to be glycosylated themselves for full activity. N-glycans and glycosyltransferases play roles in the pathogenesis of many diseases (including cancers), so studies on glycosyltransferases may contribute to the development of new therapy methods and novel glycoengineered enzymes with improved properties. In this review, we focus on the role of N-glycosylation in the activity of glycosyltransferases and attempt to summarize all available data about this phenomenon.

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells.

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

SEQUENCE SLIDER: expanding polyalanine fragments for phasing with multiple side-chain hypotheses.

Fragment-based molecular-replacement methods can solve a macromolecular structure quasi-ab initio. ARCIMBOLDO, using a common secondary-structure or tertiary-structure template or a library of folds, locates these with Phaser and reveals the rest of the structure by density modification and autotracing in SHELXE. The latter stage is challenging when dealing with diffraction data at lower resolution, low solvent content, high ß-sheet composition or situations in which the initial fragments represent a low fraction of the total scattering or where their accuracy is low. SEQUENCE SLIDER aims to overcome these complications by extending the initial polyalanine fragment with side chains in a multisolution framework. Its use is illustrated on test cases and previously unknown structures. The selection and order of fragments to be extended follows the decrease in log-likelihood gain (LLG) calculated with Phaser upon the omission of each single fragment. When the starting substructure is derived from a remote homolog, sequence assignment to fragments is restricted by the original alignment. Otherwise, the secondary-structure prediction is matched to that found in fragments and traces. Sequence hypotheses are trialled in a brute-force approach through side-chain building and refinement. Scoring the refined models through their LLG in Phaser may allow discrimination of the correct sequence or filter the best partial structures for further density modification and autotracing. The default limits for the number of models to pursue are hardware dependent. In its most economic implementation, suitable for a single laptop, the main-chain trace is extended as polyserine rather than trialling models with different sequence assignments, which requires a grid or multicore machine. SEQUENCE SLIDER has been instrumental in solving two novel structures: that of MltC from 2.7 Šresolution data and that of a pneumococcal lipoprotein with 638 residues and 35% solvent content.

3D Structure and Function of Glycosyltransferases Involved in N-glycan Maturation.

Glycosylation is the most ubiquitous post-translational modification in eukaryotes. N-glycan is attached to nascent glycoproteins and is processed and matured by various glycosidases and glycosyltransferases during protein transport. Genetic and biochemical studies have demonstrated that alternations of the N-glycan structure play crucial roles in various physiological and pathological events including progression of cancer, diabetes, and Alzheimer's disease. In particular, the formation of N-glycan branches regulates the functions of target glycoprotein, which are catalyzed by specific N-acetylglucosaminyltransferases (GnTs) such as GnT-III, GnT-IVs, GnT-V, and GnT-IX, and a fucosyltransferase, FUT8s. Although the 3D structures of all enzymes have not been solved to date, recent progress in structural analysis of these glycosyltransferases has provided insights into substrate recognition and catalytic reaction mechanisms. In this review, we discuss the biological significance and structure-function relationships of these enzymes.

Functional analysis of glycosylation using Drosophila melanogaster.

The glycosylation of proteins and lipids has various essential roles in a diverse range of biological processes, including embryogenesis, organ development, neurogenesis, maintenance of homeostasis, immune response, and tumorigenesis. Drosophila melanogaster is one of the representative multicellular model organisms, which have many useful genetic manipulation tools; it is used in developmental biology as well as classical and molecular genetics. Glycobiology is not an exception and many studies using Drosophila have been performed in this field to clarify novel functions of glycans. Recently, genome-wide screening and functional analyses were performed in whole body, wings, eyes, neuromuscular junctions, and immune organs. Furthermore, detailed studies with Drosophila mutants of glycosyltransferases, nucleotide sugar transporters, and glycosidases revealed novel functions of N-linked glycans, glycosaminoglycans, glycolipids, and O-linked glycans including mucin type O-glycan, O-Fuc, O-Man, and O-GlcNAc. As many of these functions are common between Drosophila and humans, these mutants represent good models for human disease. In this review, recent studies of glycan functions using Drosophila are summarized.

Enzymatic O-Glycosylation of Etoposide Aglycone by Exploration of the Substrate Promiscuity for Glycosyltransferases.

The 4-O-ß-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.

On the Catalytic Activity of a GT1 Family Glycosyltransferase from Streptomyces venezuelae ISP5230.

GT1 family glycosyltansferase, Sv0189, from Streptomyces venezuelae ISP5230 (ATCC 10721) was characterized. The recombinantly produced protein Sv0189 possessed UDP-glycosyltransferase activity. Screening, using an assay employing unnatural nitrophenyl glycosides as activated donors, resulted in the discovery of a broad substrate scope with respect to both acceptor molecules and donor sugars. In addition to polyphenols, including anthraquinones, simple aromatics containing primary or secondary alcohols, a variety of complex natural products and synthetic drugs were glucosylated or xylosylated by Sv0189. Regioselectivity was established through the isolation and characterization of glucosylated products. Sv0189 and homologous proteins are widely distributed among Streptomyces species, and their apparent substrate promiscuity reveals potential for their development as biocatalysts for glycodiversification.

In vitro characterization of MitE and MitB: Formation of N-acetylglucosaminyl-3-amino-5-hydroxybenzoyl-MmcB as a key intermediate in the biosynthesis of antitumor antibiotic mitomycins.

Mitomycins, produced by several Streptomyces strains, are potent anticancer antibiotics that comprise an aziridine ring fused to a tricyclic mitosane core. Mitomycins have remarkable ability to crosslink DNA with high efficiency. Despite long clinical history of mitomycin C, the biosynthesis of mitomycins, especially mitosane core formation, remains unknown. Here, we report in vitro characterization of three proteins, MmcB (acyl carrier protein), MitE (acyl AMP ligase), and MitB (glycosyltransferase) involved in mitosane core formation. We show that 3-amino-5-hydroxybenzoic acid (AHBA) is first loaded onto MmcB by MitE at the expense of ATP. MitB then catalyzes glycosylation of AHBA-MmcB with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to generate a key intermediate, GlcNAc-AHBA-MmcB, which contains all carbon and nitrogen atoms of the mitosane core. These results provide important insight into mitomycin biosynthesis.