O antigen biosynthesis in Gram-negative bacteria: Characterization of alpha1,4-glucosyltransferase WclY from Escherichia coli O117 and development of glycosyltransferase inhibitors.
Bibliographic record
Abstract
Gram-negative bacteria have a polysaccharide coat (O antigen) consisting of repeating units of oligosaccharides that is assembled by glycosyltransferases. Many of these bacteria are associated with disease, and the polysaccharides are functionally important for immune responses. In light of increasing antibiotic resistance it is critical to develop new antibacterial strategies and gain knowledge of the mechanisms by which bacteria assemble their virulence factors. We hypothesize that inhibition of the glycosyltransferases responsible for O antigen synthesis in pathogenic Gram-negative bacteria can lead to the attenuation of bacteria’s virulence. The O antigens in O117 and O107 serotypes of Escherichia coli, differ by a single sugar residue. The glucose (Glc) residue found in the O antigen of E. coli O117 [D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAc]n is replaced by N-acetylglucosamine (GlcNAc) in the O antigen of E. coli O107 (GalNAc is N-acetylgalactosamine, Rha is Rhamnose, Gal is galactose). The glycosyltransferases that create the Glc/GlcNAcα1-4Gal linkage are WclY(O117) and WclY(O107), and differ in only 3 amino acid residues. We have expressed WclY(O117) in a soluble and active form in the Lemo21 (DE3) expression system. We developed an activity assay for Glc-transferase WclY using a synthetic analog of its natural acceptor substrate and subsequently characterized the enzyme. WclY(O117) is a retaining Glc-transferase (sugar of the donor substrate retains α/β stereochemistry post-transfer) of the Carbohydrate Active Enzymes (CAZy) GT4 family; it has a strict substrate specificity requiring Galβ1-3GalNAcα-diphosphate lipid as the acceptor. In contrast, the donor substrate specificity is more relaxed. The structures of reaction products were confirmed using Electrospray Ionization-Negative ion mode Mass Spectrometry as well as Nuclear Magnetic Resonance Spectroscopy. Mutants were expressed to provide insight into the catalytic mechanism of WclY. Mutants transferred sugars from UDP-Glc, UDP-GlcNAc, UDP-Gal or UDP-GalNAc at varying levels. By mutating Arg194 we were able to convert the Glc-transferase to a GlcNAc-transferase. Protein modeling confirmed the role of Arg194 and other amino acids in the catalytic activity of WclY. Characterization of the enzyme has opened the door for testing of compounds with the potential to act as antimicrobial and antiseptic agents.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".