Characterization of the UDP-Glc/GlcNAc 4-epimerase AglW from the archaeon Methanococcus maripaludis using glycosyltransferase coupled assays
Bibliographic record
Abstract
Archaea are ubiquitous single-celled microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis, a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with the N-linked tetrasaccharide: 4-deoxy-5-O-Me-ManNAc β1-4 ManNAc3NAmA6Thr β1-4 GlcNAc3NAcA β1-3 GalNAc β-Asn, where the sugar 4-deoxy-5-O-Me-ManNAc is found exclusively in this species. The attachment of the N-linked tetrasaccharide to archaellins is required for proper motility of M. maripaludis. The aglW gene encoding the putative UDP-Glc/GlcNAc 4-epimerase AglW plays a key role in the biosynthesis of the unique terminal sugar residue found in the N-linked tetrasaccharide. The goal of this study was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop glycosyltransferase (GT) coupled assays to determine its substrate specificity and properties. We carried out coupled assays using UDP-Gal, UDP-Glc, UDP-GlcNAc, UDP-GalNAc and GlcNAc/GalNAc-diphosphate – lipid as substrates. We established that AglW is active without the addition of divalent metal ions or NAD+ and has a broad specificity towards UDP-sugars. We also showed that tyrosine-151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The GT-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases. The results of this work provide insight into the synthesis of complex N-glycans in M. maripaludis.
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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) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.001 | 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".