Lyme Disease Enolpyruvyl-UDP-GlcNAc Synthase: Fosfomycin-Resistant MurA from <i>Borrelia burgdorferi</i>, a Fosfomycin-Sensitive Mutant, and the Catalytic Role of the Active Site Asp
Bibliographic record
Abstract
MurAs (enolpyruvyl-UDP-GlcNAc synthases) from pathogenic bacteria such as Borrelia burgdorferi (Lyme disease) and tuberculosis are fosfomycin resistant because an Asp-for-Cys substitution prevents them from being alkylated by this epoxide antibiotic. Previous attempts to characterize naturally Asp-containing MurAs have resulted in no protein or no activity. We have expressed and characterized His-tagged Lyme disease MurA (Bb_MurA(H6)). The protein was most soluble at high salt concentrations but maximally active around physiological ionic strength. The steady-state kinetic parameters at pH 7 were k(cat) = 1.07 ± 0.03 s(-1), K(M,PEP) = 89 ± 12 μM, and K(M,UDP-GlcNAc) = 45 ± 7 μM. Mutating the active site Asp to Cys, D116C, caused a 21-fold decrease in k(cat) and rendered the enzyme fosfomycin sensitive. The pH profile of k(cat) was bell-shaped and centered around pH 5.3 for Bb_MurA(H6), with pK(a1) = 3.8 ± 0.2 and pK(a2) = 7.4 ± 0.2. There was little change in pK(a1) with the D116C mutant, 3.5 ± 0.3, but pK(a2) shifted to >11. This demonstrated that the pK(a2) of 7.4 was due to D116, almost 3 pH units above an unperturbed carboxylate, and that it must be protonated for activity. This supports D116's proposed role as a general acid/base catalyst. As fosfomycin does not react with simple thiols, nor most protein thiols, the reactivity of D116C with fosfomycin, combined with the strongly perturbed pK(a2) for D116, strongly implies an unusual active site environment and a chemical role in catalysis for Asp/Cys. There is also good evidence for C115 having a role in product release. Both roles may be operative for both Asp- and Cys-containing MurAs.
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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 | 0.001 |
| 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 | 0.001 |
| 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 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".