Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids
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Bibliographic record
Abstract
Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis. Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.
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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 | 0.000 |
| 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 | 0.001 | 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 it