Biosynthesis of di-N-acetylated mannuronic acid in Pseudomonas aeruginosa and Bordetella pertussis
Bibliographic record
Abstract
Both 'Pseudomonas aeruginosa' and 'Bordetella pertussis ' produce UDP-2,3-diacetamido-2,3-dideoxy-[alpha]-D-mannuronic acid (UDP-ManNAc3NAcA) as a precursor required for incorporation into lipopolysaccharide (LPS). A pathway for the biosynthesis of UDP-ManNAc3NAcA was proposed and requires the products of five genes from 'P. aeruginosa: wbpA, wbpB, wbpE, wbpD,' and 'wbpI'. Homologous protein products were identified in 'B. pertussis', and were investigated through a genetic complementation strategy. Analytical separation of the LPS was used to score the ability of plasmid-borne genes from 'B. pertussis' to complement individual 'wbp' gene knockouts in 'P. aeruginosa'. In this manner, 'wbpO, wlbA, wlbC, wlbB,' and 'wlbD' of 'B. pertussis' were shown to be functionally identical to 'wbpA, wbpB, wbpE, wbpD,' and ' wbpI', respectively. Further biochemical analysis of two versions of 'B. pertussis' WbpO showed that that both are UDP-' N'-acetylglucosamine 6-dehydrogenases, like WbpA, but with additional 6-dehydrogenase activity against UDP-'N'-acetylgalactosamine and UDP-glucose. Using capillary electrophoresis and mass spectrometry, WbpB and WbpE were shown to work in concert on UDP-'N'-acetylglucosaminuronic acid (UDP-GlcNAcA) to produce UDP-2-acetamido-3-keto-2,3-dideoxyglucuronic acid (UDP-3-keto-GlcNAcA) and UDP-2-acetamido-3-amino-2,3-dideoxyglucuronic acid (UDP-GlcNAc3NA), respectively. Purified UDP-GlcNAc3NA was characterized by NMR. This gave evidence to support the classification of WbpB as a UDP-GlcNAcA 3-dehydrogenase and WbpE as a UDP-3-keto-GlcNAcA transaminase. Furthermore, WbpB was shown to require NAD+ as a cofactor and MgCl 2 for full activity; WbpE was shown to require pyridoxal phosphate and L-glutamate. WbpD, demonstrated to be a UDP-GlcNAc3NA 'N'-acetyltransferase, then produces UDP-2,3-diacetamido-2,3-dideoxy-[alpha]-D-glucuronic acid (UDP-GlcNAc3NAcA), which was verified through NMR spectrometry. The final reaction uses UDP-GlcNAc3NAcA in a 2-epimerization catalyzed by WbpI of ' P. aeruginosa' or WlbD of 'B. pertussis'. The epimerase reaction was followed by live-reaction NMR, which demonstrated that the enzymes do not require any cofactors and reach a maximum of about 15% conversion. This research has presented the first evidence for the functions of WbpB, WbpE, WbpD, WbpI and the 'B. pertussis' homologs WlbA, WlbC, WlbB, and WlbD, respectively. Using overexpressed and purified recombinant enzymes, the entire UDP-ManNAc3NAcA biosynthesis pathway of 'P. aeruginosa ' was reconstituted 'in vitro', providing strong evidence that the pathway proposed for UDP-ManNAc3NAcA biosynthesis is correct in both ' P. aeruginosa' and 'B. pertussis'.
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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) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 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".