Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in <i>Campylobacter jejuni</i> 81116 <sup>†</sup>
Why this work is in the frame
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Bibliographic record
Abstract
The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.
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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) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 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 it