Review

Authors: Robyn Meech, Dong Gui Hu, Ross A McKinnon...

UDP-glycosyltransferases (UGTs) catalyze the covalent addition of sugars to a broad range of lipophilic molecules. This biotransformation plays a critical role in elimination of a broad range of exogenous chemicals and by-products of endogenous metabolism, and also controls the levels and distribution of many endogenous signaling molecules. In mammals, the superfamily comprises four families: UGT1, UGT2, UGT3, and UGT8. UGT1 and UGT2 enzymes have important roles in pharmacology and toxicology including contributing to interindividual differences in drug disposition as well as to cancer risk. These UGTs are highly expressed in organs of detoxification (e.g., liver, kidney, intestine) and can be induced by pathways that sense demand for detoxification and for modulation of endobiotic signaling molecules. The functions of the UGT3 and UGT8 family enzymes have only been characterized relatively recently; these enzymes show different UDP-sugar preferences to that of UGT1 and UGT2 enzymes, and to date, their contributions to drug metabolism appear to be relatively minor. This review summarizes and provides critical analysis of the current state of research into all four families of UGT enzymes. Key areas discussed include the roles of UGTs in drug metabolism, cancer risk, and regulation of signaling, as well as the transcriptional and posttranscriptional control of UGT expression and function. The latter part of this review provides an in-depth analysis of the known and predicted functions of UGT3 and UGT8 enzymes, focused on their likely roles in modulation of levels of endogenous signaling pathways.

Topics: Animals; Gene Expression Regulation, Enzymologic; Glycosyltransferases; Mammals; Multigene Family; Signal Transduction

PubMed: 30724669
DOI: 10.1152/physrev.00058.2017

Review

Authors: Eric Sauvage, Frédéric Kerff, Mohammed Terrak...

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

Topics: Bacteria; Bacterial Proteins; Glycosyltransferases; Multienzyme Complexes; Penicillin-Binding Proteins; Peptide Hydrolases; Peptidoglycan; Peptidoglycan Glycosyltransferase; Peptidyl Transferases; Protein Structure, Tertiary; beta-Lactamases; beta-Lactams

PubMed: 18266856
DOI: 10.1111/j.1574-6976.2008.00105.x

Authors: Brandi L Cantarel, Pedro M Coutinho, Corinne Rancurel...

The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families. These families are created based on experimentally characterized proteins and are populated by sequences from public databases with significant similarity. Protein biochemical information is continuously curated based on the available literature and structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification (i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties. This resource has been available for over 10 years to the scientific community, contributing to information dissemination and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/.

Topics: Carbohydrate Metabolism; Carrier Proteins; Databases, Protein; Esterases; Glycoconjugates; Glycomics; Glycoside Hydrolases; Glycosyltransferases; Polysaccharide-Lyases

PubMed: 18838391
DOI: 10.1093/nar/gkn663

Authors: Victoria M Marando, Daria E Kim, Phillip J Calabretta...

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of d-arabinofuranose (d-Ara), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including . The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of d-Ara residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

Topics: Polysaccharides; Mycobacterium tuberculosis; Glycosyltransferases; Humans

PubMed: 34606245
DOI: 10.1021/jacs.1c07430

Authors: Marcelo E Guerin

Glycosyltransferases (GTs) attach sugar molecules to a broad range of acceptors, generating a remarkable amount of structural diversity in biological systems. GTs are classified as either "retaining" or "inverting" enzymes. Most retaining GTs typically use an Si mechanism. In a recent article in the JBC, Doyle et al. demonstrate a covalent intermediate in the dual-module KpsC GT (GT107) supporting a double displacement mechanism.

Topics: Glycosyltransferases

PubMed: 37394002
DOI: 10.1016/j.jbc.2023.105006

Review

Authors: Carolina Ortiz-Cordero, Karim Azzag, Rita C R Perlingeiro...

Fukutin-related protein (FKRP) is a glycosyltransferase involved in the functional glycosylation of α-dystroglycan (DG), a key component in the link between the cytoskeleton and the extracellular matrix (ECM). Mutations in FKRP lead to dystroglycanopathies with broad severity, including limb-girdle and congenital muscular dystrophy. Studies over the past 5 years have elucidated the function of FKRP, which has expanded the number of therapeutic opportunities for patients carrying FKRP mutations. These include small molecules, gene delivery, and cell therapy. Here we summarize recent findings on the function of FKRP and describe available models for studying diseases and testing therapeutics. Lastly, we highlight preclinical studies that hold potential for the treatment of FKRP-associated dystroglycanopathies.

Topics: Dystroglycans; Glycosylation; Glycosyltransferases; Humans; Muscular Dystrophies; Pentosyltransferases

PubMed: 33272829
DOI: 10.1016/j.tcb.2020.11.003

Review

Authors: Thomas Louveau, Anne Osbourn

Glycosylation plays a major role in the structural diversification of plant natural products. It influences the properties of molecules by modifying the reactivity and solubility of the corresponding aglycones, so influencing cellular localization and bioactivity. Glycosylation of plant natural products is usually carried out by uridine diphosphate(UDP)-dependent glycosyltransferases (UGTs) belonging to the carbohydrate-active enzyme glycosyltransferase 1 (GT1) family. These enzymes transfer sugars from UDP-activated sugar moieties to small hydrophobic acceptor molecules. Plant GT1s generally show high specificity for their sugar donors and recognize a single UDP sugar as their substrate. In contrast, they are generally promiscuous with regard to acceptors, making them attractive biotechnological tools for small molecule glycodiversification. Although microbial hosts have traditionally been used for heterologous engineering of plant-derived glycosides, transient plant expression technology offers a potentially disruptive platform for rapid characterization of new plant glycosyltransferases and biosynthesis of complex glycosides.

Topics: Biocatalysis; Biological Products; Glycosylation; Glycosyltransferases; Phylogeny; Plants; Protein Conformation; Uridine Diphosphate

PubMed: 31235546
DOI: 10.1101/cshperspect.a034744

Authors: Dietlind L Gerloff, Elena I Ilina, Camille Cialini...

A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022)..

Topics: Protein Engineering; Biological Assay; Computational Biology; Glycosyltransferases; Mutation

PubMed: 36528856
DOI: 10.1016/j.xpro.2022.101905

Authors: Fabienne Gutacker, Yvonne-Isolde Schmidt-Bohli, Tina Strobel...

Glycosyltransferases are important enzymes which are often used as tools to generate novel natural products. In this study, we describe the identification and characterization of an inverting - and -glycosyltransferase from NRRL2338. When feeding experiments with 1,4-diaminoanthraquinone in were performed, the formation of new compounds (U3G and U3DG) was observed by HPLC-MS. Structure elucidation by NMR revealed that U3G consists of two compounds, -α-glucosyl-1,4-diaminoanthraquinone and -β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a ,-diglucosyl-1,4-diaminoanthraquinone. In order to find the responsible glycosyltransferase, gene deletion experiments were performed and we identified the glycosyltransferase Sace_3599, which belongs to the CAZy family 1. When J1074, containing the dTDP-d-glucose synthase gene and the plasmid pUWL-A-, was used as host, U3 was converted to the same compounds. Protein production in and purification of Sace_3599 was carried out. The enzyme showed glycosyl hydrolase activity and was able to produce mono- and di--glycosylated products in vitro. When UDP-α-d-glucose was used as a sugar donor, U3 was stereoselective converted to -β-glucosyl-1,4-diaminoanthraquinone and ,-diglucosyl-1,4-diaminoanthraquinone. The use of 1,4-dihydroxyanthraquinone as a substrate in in vitro experiments also led to the formation of mono-glucosylated and di-glucosylated products, but in lower amounts. Overall, we identified and characterized a novel glycosyltransferase which shows glycohydrolase activity and the ability to glycosylate "drug like" structures forming - and -glycosidic bonds.

Topics: Amino Acid Sequence; Anthraquinones; Bacterial Proteins; Genome, Bacterial; Glycosylation; Glycosyltransferases; Saccharopolyspora; Sequence Homology

PubMed: 32727097
DOI: 10.3390/molecules25153400

Review

Authors: Beata Filipek-Górniok, Judith Habicher, Johan Ledin...

The biosynthesis of heparan sulfate (HS) proteoglycans occurs in the Golgi compartment of cells and will determine the sulfation pattern of HS chains, which in turn will have a large impact on the biological activity of the proteoglycans. Earlier studies in mice have demonstrated the importance of HS for embryonic development. In this review, the enzymes participating in zebrafish HS biosynthesis, along with a description of enzyme mutants available for functional studies, are presented. The consequences of the zebrafish genome duplication and maternal transcript contribution are briefly discussed as are the possibilities of CRISPR/Cas9 methodologies to use the zebrafish model system for studies of biosynthesis as well as proteoglycan biology.

Topics: Animals; Biosynthetic Pathways; CRISPR-Cas Systems; Glycosyltransferases; Heparitin Sulfate; Mutation; Sulfotransferases; Zebrafish; Zebrafish Proteins

PubMed: 33216642
DOI: 10.1369/0022155420973980