Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates
Bibliographic record
Abstract
Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O -demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon–carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O -demethylation of para -methoxylated benzoates ( p -MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity ( K d = 3.8 ± 0.6 μM) and apparent specificity ( k cat / K M = 20,000 ± 3000 M -1 s -1 ) for p- MBA. The enzyme also O -demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p- ethylbenzoate even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p -MBA, p- ethylbenzoate, and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates' carboxylate: Ser87, Ser237, and Arg84. Substitution of these residues resulted in lower affinity and O -demethylation activity on p -MBA as well as increased affinity for the acetyl analog, p- methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O -demethylation of an aldehyde analog of p -MBA, p -methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237, and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.
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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 | 0.001 | 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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| 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 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".