P450cam (CYP101A1) Mutants for Endosulfan Degradation: Complete Dehalogenation of Endosulfan Diol and Related Compounds
Bibliographic record
Abstract
Abstract Cytochrome P450cam (CYP101A1) from the soil bacterium Pseudomonas putida oxidizes camphor regio- and stereoselectively at the 5-position, to give 5-exo-hydroxycamphor. Previously, we randomly mutated P450cam and selected seven mutants on the bicyclic polychlorinated insecticide and persistent organic pollutant, endosulfan (ES). Here we describe the activity of the seven mutants of P450cam with ES diol, ES, ES lactone, ES ether, ES sulfate, and heptachlor. ES diol, ES lactone, ES ether, and ES sulfate are persistent biotransformation products of ES in the environment. They all have the hexachlorinated norbornene (bicyclo[2.2.1] hept-2-ene) moiety intact. The P450cam mutants (and to a small extent the wild type) convert these substrates to substituted ortho-quinones, which we detected using 4-aminoantipyrine (4-AAP) in spectrophotometric assays. Here we have studied dehalogenation of ES and related compounds catalyzed by the endosulfan–selected P450cam mutants, using in vitro kinetics, chloride release assays, and 13C labeled endosulfan diol. We found that mutants ES7 (V247F/D297N/K314E) and ES6 (G120S) were significantly more active towards ES diol than the WT. On average, close to six Cl- ions are released per aromatic product detected upon turnover of ES diol. Finally, product isolation from reactions with non-labeled and 13C labeled ES diol confirmed that substituted catechols formed. Based on these findings, we propose that dehalogenagion begins with the oxidation of the ES double bond on the norbornene system, proceeds with eliminating six chloride ions and loss of the bridge as CO2 to furnish an ortho-quinone, which can be reduced non-enzymatically to the corresponding catechol.
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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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".