Biology subjects

Arbour, C. A.

Publications and source records attributed to Arbour, C. A..

4 recordsLinked to original sources

bioRxiv2026

Glycoconjugate diversification in Campylobacter concisus is determined by two glycosyltransferases

Bacterial glycoconjugates are structurally diverse, with enormous variation in sugar identity, modifications and linkages. Glycoconjugates play key roles in numerous cell functions, acting as the primary interface with the environment. Asparagine (N)-linked glycosylation has been extensively studied in the pathogenic Campylobacter genus, due to the availability of numerous genome sequences and the highly conserved pathway logic, despite the final N-linked glycan product diversity. We recently reported on a partitioning of N-linked glycan structures between the Campylobacter species, focused on the inclusion of a C6-carboxyl-sugar in the third position of the growing glycan in Campylobacter concisus. However, at the time, the final glycan was not fully defined. Here, we identify the final glycan product in C. concisus, demonstrating surprising substrate promiscuity of the GT-A enzyme, PglI, which adds the penultimate sugar, and uncover a previously uncharacterized enzyme (GT-25) that unexpectedly adds the final sugar to complete the heptasaccharide product. Through a detailed study of these two C. concisus pathway enzymes, the intermediate and final glycans were defined, with determination of linkage positions of major and minor isomeric products following each glycan addition, through high-resolution electronic excitation dissociation tandem mass spectrometry. These findings on the Group II C. concisus N-linked glycan highlight the diversification of the glycan and the utilization, at the non-reducing end, of GlcNAc over GalNAc, which is dominant in the Group I species.

biochemistry↗

bioRxiv2025

Correlating membrane-protein dynamics with function: Integrating bioinformatics, molecular dynamics, and single-molecule FRET

We present a strategy that deploys structural bioinformatics, molecular simulation, and single-molecule FRET microscopy for observing the ligand-dependent conformational dynamics of integral membrane proteins in situ. We focus on representative members of the small monotopic phosphoglycosyl transferase (SmPGT) superfamily, which catalyze transfer of a phosphosugar from a soluble nucleotide-sugar donor to a membrane-embedded polyprenol phosphate acceptor in the initiating step of glycoconjugate biosynthesis in prokaryotes. Substrate-specific structural features were identified across the superfamily and correlated with ligand-dependent conformational dynamics in all-atom simulations. To experimentally validate the role of this motion in ligand binding, we developed a platform to monitor intra-molecular protein dynamics in a native-like lipid environment. The presented approach incorporates selective cysteine protein labeling and non-canonical amino acid mutagenesis with bicyclononyne-tetrazine click chemistry to assemble dual-labeled variants of PglC, the initiating enzyme of the N-linked protein glycosylation pathway from Campylobacter jejuni. The modified proteins are then solubilized into styrene maleic acid liponanoparticles (SMALPs) to maintain an in situ membrane environment. The conformational changes of PglC upon inhibitor binding are diagnostic of inhibitor potency. The single-molecule FRET-SMALP strategy can be adapted to investigate protein dynamics across the superfamily of SmPGTs with different substrate selectivity where structure prediction and molecular dynamics support significant conformational changes upon ligand binding. Broader ImpactMembrane protein structure-function relationships are critical for understanding fundamental biological processes and for the development of small-molecule drug treatments. Bacterial glycoconjugate biosynthesis pathways are a promising target for strain-specific antibiotics and exemplify the challenges of characterizing biomolecular systems that depend on highly specific protein, lipid, and carbohydrate chemistries. We integrate molecular simulation, structural bioinformatics, and single-molecule FRET to elucidate details of small-molecule binding to the PGT superfamily.

biochemistry↗

bioRxiv2024

Functionally Validated Proteome-Wide Bioinformatic Annotation of the Monotopic Phosphoglycosyl Transferase Family

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network (SSN) analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in a bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives. SignificanceUncovering the function and specificity of enzymes responsible for glycoconjugate biosynthesis traditionally requires a multi-faceted and individually curated approach. This is especially true for bacterial glycoconjugates due to greater monosaccharide diversity and a paucity of established structural information. Here we leverage bioinformatic and in-vitro tools to predict and validate substrate specificity for a unique, exclusively bacterial family of enzymes responsible for the first step in many of these glycan assembly pathways. We further show that this platform is suitable for enhanced functional annotation and inhibitor testing, paving the way for the development of urgently needed antibiotics.

biochemistry↗

bioRxiv2023

Defining Early Steps in B. subtilis Biofilm Biosynthesis

The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the exopolysaccharide of B. subtilis remains unclear and incomplete. This report presents synergistic biochemical and genetic studies built from a foundation of comparative sequence analyses targeted at elucidating the activities of the first two membrane-committed steps in the exopolysaccharide biosynthetic pathway. By taking this approach, we determined the nucleotide sugar donor and lipid-linked acceptor substrates for the first two enzymes in the B. subtilis biofilm exopolysaccharide biosynthetic pathway. EpsL catalyzes the first phosphoglycosyl transferase step using UDP-di-N-acetyl bacillosamine as phospho-sugar donor. EpsD is a GT-B fold glycosyl transferase that facilitates the second step in the pathway that utilizes the product of EpsL as an acceptor substrate and UDP-N-acetyl glucosamine as the sugar donor. Thus, the study defines the first two monosaccharides at the reducing end of the growing exopolysaccharide unit. In doing so we provide the first evidence of the presence of bacillosamine in an exopolysaccharide synthesized by a Gram-positive bacterium. IMPORTANCEBiofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide synthesis pathway. Together our studies and approaches provide the foundation for the sequential characterization of the steps in exopolysaccharide biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenol diphosphate-linked glycan substrates.

microbiology↗