Glycosyltransferases Involved in Biosynthesis of the Outer Core Region of Escherichia coli Lipopolysaccharides Exhibit Broader Substrate Specificities Than Is Predicted from Lipopolysaccharide Structures
Bibliographic record
Abstract
The waaJ, waaT, and waaR genes encode alpha-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS alpha-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS alpha-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in k(cat) and corresponding increases in K(m)(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His(6)-WaaT, His(6)-WaaJ showed a significantly decreased k(cat) and increased K(m)(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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 itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), 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".