A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes

Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD+ generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima.National Institutes of Health (U.S.) (GM-039334

Priming and processing glycosyltransferases in bacterial N-linked glycosylation pathways

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.Bacterial cell surfaces prominently feature a variety of complex glycoconjugates. Although these structures are very diverse, the biosynthesis of these glycoconjugates shares common themes. In particular, a priming glycosyltransferase or phosphoglycosyltransferase (PGT) initiates the biosynthesis of glycans by transferring a Cl'-phosphosugar onto a polyprenol phosphate substrate. The membrane-bound polyprenol diphosphosugar product is then further elaborated by additional glycosyltransferases, flipped across the inner bacterial membrane, and ultimately used as glycosyl donor substrate in transfer to a final acceptor substrate, which, is a lipid, a protein or another glycan. This thesis focuses on the priming GTs that catalyze the first step in glycoconjugate biosynthesis. This class of enzymes is structurally diverse and includes several distinct families of enzymes with different structures and membrane topologies. PglC, the smallest and structurally, the simplest PGT found in Campylobacter jejuni, represents a tractable model with which to investigate this class of enzymes. First, efforts to structurally characterize PglC are discussed, with a focus on the challenges associated with expressing, purifying and characterizing integral membrane proteins. The next section of this thesis presents the functional characterization of PglC using a variety of biochemical techniques. In addition, bioinformatics analysis of PglC and related PGT families was employed to provide insight into critical residues required for catalytic activity. To complement this, a predicted structure of PgIC was developed, based on the wealth of information contained in the sequences of homologs of PglC. Kinetic analysis of PglC was used to investigate the mechanism of the PGT reaction. Together, these studies aided in the design and evaluation of inhibitors of PgIC, inspired by the nucleoside antibiotics tunicamycin and mureidomycin. In the final chapter, the enzymes in the N-linked protein glycosylation pathway in Campylobacter jejuni were evaluated for their tolerance for azide-modified UDP-sugar substrates. In vitro experiments were employed to investigate the potential of these enzymes for executing the chemoenzymatic synthesis of the C. jejuni glycan with azide-modified sugars at discrete targeted positions. Attempts to metabolically label C. jejuni with azide-modified sugars for in vivo incorporation into the N-glycan are also presented.by Vinita Lukose.Ph. D

Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface

Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes.National Institutes of Health (Grant GM-039334

A Modular Approach to Phosphoglycosyltransferase Inhibitors Inspired by Nucleoside Antibiotics

Phosphoglycosyltransferases (PGTs) represent "gatekeeper" enzymes in complex glycan assembly pathways by catalyzing transfer of a phosphosugar from an activated nucleotide diphosphosugar to a membrane-resident polyprenol phosphate. The unique structures of selected nucleoside antibiotics, such as tunicamycin and mureidomycin A, which are known to inhibit comparable biochemical transformations, are exploited as the foundation for the development of modular synthetic inhibitors of PGTs. Herein we present the design, synthesis, and biochemical evaluation of two readily manipulatable modular scaffolds as inhibitors of monotopic bacterial PGTs. Selected compounds show IC50 values down to the 40 μM range, thereby serving as lead compounds for future development of selective and effective inhibitors of diverse PGTs of biological and medicinal interest

Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface

Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes

Bacterial phosphoglycosyl transferases:initiators of glycan biosynthesis at the membrane interface

Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes

Chemoenzymatic Assembly of Bacterial Glycoconjugates for Site-Specific Orthogonal Labeling

The cell surfaces of bacteria are replete with diverse glycoconjugates that play pivotal roles in determining how bacteria interact with the environment and the hosts that they colonize. Studies to advance our understanding of these interactions rely on the availability of chemically defined glycoconjugates that can be selectively modified under orthogonal reaction conditions to serve as discrete ligands to probe biological interactions, in displayed arrays and as imaging agents. Herein, enzymes in the N-linked protein glycosylation (Pgl) pathway of Campylobacter jejuni are evaluated for their tolerance for azide-modified UDP-sugar substrates, including derivatives of 2,4-diacetamidobacillosamine and N-acetylgalactosamine. In vitro analyses reveal that chemoenzymatic approaches are useful for the preparation of undecaprenol diphosphate-linked glycans and glycopeptides with site-specific introduction of azide functionality for orthogonal labeling at three specific sites in the heptasaccharide glycan. The uniquely modified glycoconjugates represent valuable tools for investigating the roles of C. jejuni cell surface glycoconjugates in host pathogen interactionsNational Institutes of Health (U.S.) (Grant GM-039334)Natural Sciences and Engineering Research Council of Canada (Fellowship

Conservation and Covariance in Small Bacterial Phosphoglycosyltransferases Identify the Functional Catalytic Core

Phosphoglycosyltransferases (PGTs) catalyze the transfer of a C1′-phosphosugar from a soluble sugar nucleotide diphosphate to a polyprenol phosphate. These enzymes act at the membrane interface, forming the first membrane-associated intermediates in the biosynthesis of cell-surface glycans and glycoconjugates, including glycoproteins, glycolipids, and the peptidoglycan in bacteria. PGTs vary greatly in both their membrane topologies and their substrate preferences. PGTs, such as MraY and WecA, are polytopic, while other families of uniquely prokaryotic enzymes have only a single predicted transmembrane helix. PglC, a PGT involved in the biosynthesis of N-linked glycoproteins in the enteropathogen Campylobacter jejuni, is representative of one of the structurally most simple members of the diverse family of small bacterial PGT enzymes. Herein, we apply bioinformatics and covariance-weighted distance constraints in geometry- and homology-based model building, together with mutational analysis, to investigate monotopic PGTs. The pool of 15000 sequences that are analyzed include the PglC-like enzymes, as well as sequences from two other related PGTs that contain a “PglC-like” domain embedded in their larger structures (namely, the bifunctional PglB family, typified by PglB from Neisseria gonorrheae, and WbaP-like enzymes, typified by WbaP from Salmonella enterica). Including these two subfamilies of PGTs in the analysis highlights key residues conserved across all three families of small bacterial PGTs. Mutagenesis analysis of these conserved residues provides further information about the essentiality of many of these residues in catalysis. Construction of a structural model of the cytosolic globular domain utilizing three-dimensional distance constraints, provided by conservation covariance analysis, provides additional insight into the catalytic core of these families of small bacterial PGT enzymes.National Institutes of Health (U.S.) (NIH Grant Number: R21 AI101807)National Institutes of Health (U.S.) (NIH Grant Number: GM039334)National Institutes of Health (U.S.) (NIH Grant Number: R01 GM064700)National Institutes of Health (U.S.) (NIH Grant Number: R01 GM 061867