Indigo Formation as a Predictor of Substrate Promiscuity in Cytochrome P450 BM3 Libraries
Bibliographic record
Abstract
Cytochrome P450 BM3 natively exhibits substrate promiscuity, mainly in hydroxylation and epoxidation reactions. Whereas directed evolution has broadened its scope of reactivity, screening for specific non-native enzymatic reactivity remains challenging, thereby limiting potential applications. In this study, we investigated the predictive potential of indigo production as an indicator of promiscuous hydroxylation in non-native aromatic compounds. Following site- saturation mutagenesis on 42 residues in the active-site region, we rapidly identified 97 previously unreported variants at 18 unique positions, significantly increasing the database of indigo-positive variants. From this expanded pool, we isolated 80 well-expressed indigo-positive variants and evaluated them alongside 46 well-expressed indigo-negative variants for their hydroxylation activity on the representative non-native aromatic compounds, anisole and naphthalene. We observed a strong correlation between indigo formation and aromatic compound hydroxylation, with 73-80% of indigo-positive variants hydroxylating these compounds, compared to 40-45% of indigo-negative variants. Notably, only indigo-positive variants showed greater than 3-fold higher activity than the wild-type P450 BM3. Furthermore, a positive correlation was obtained between promiscuous hydroxylation of anisole and naphthalene. The approach also allowed identification of new variants that hydroxylate a further aromatic compound, 4-phenyl-2-butanone, suggesting generality. Although we did not succeed in identifying the molecular basis underlying the indigo- positive phenotype, our findings demonstrate that indigo production is an effective screening tool, expediting the discovery of novel functional genes for promiscuous aromatic hydroxylation reactions. This method offers a cost-effective and efficient approach to identify and develop new biocatalysts, advancing the potential applications of P450 BM3 in scientific and industrial fields.
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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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| 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) | 0.001 | 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 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".