P450 enzymes in biocatalysis: exploration of chemical auxiliaries, macromolecular crowding, bioconjugation and oriented-immobilization
Bibliographic record
Abstract
Cytochrome P450 enzymes (CYPs or P450s) form a ubiquitous family of heme-dependent monooxygenases known mainly for their role in xenobiotic metabolism and their remarkable ability to regio- and stereoselectively oxidize inactivated C-H bonds, a feat that is difficult to achieve by chemical methods. Unfortunately, our ability to study and exploit these enzymes as in vitro biocatalysts has been limited by their low activity, low stability and poor product predictability. This thesis focuses on the study of human drug metabolizing P450 isoforms, namely CYP2E1, CYP3A4 and CYP2D6 because of their exceptional ability to accept a large variety of substrates. In Chapter 2, we demonstrate the utility of "type II ligands" as chemical auxiliaries for biocatalysis with human CYP2E1. We show that linking the chemical auxiliary nicotinate to a variety of short hydrocarbon substrates can promote their oxidation with predictable regioselectivity at the secondary aliphatic or alkenyl C-H bond furthest from the auxiliary. The origin of this selectivity was rationalized through docking studies of our auxiliary-substrate compounds with reported X-ray crystals structures of CYP2E1. These results not only confirm the general utility of the chemical auxiliary approach pioneered by our lab to direct the predictable oxidation of inactivated C-H bonds by P450 enzymes, but also provide a system with complementary regioselectivity.A short study of the effects of macromolecular crowding on the activity of human CYP3A4 and CYP2D6 is presented in Chapter 3. We found that certain crowding agents were not detrimental to enzyme activity while others had a negative effect. Moreover, certain conditions (initially tested as controls) that improved enzymatic activity were uncovered. In Chapter 4, the non-covalent oriented-immobilization of CYP3A4 via its C-terminal histidine-tag is described. We show that immobilization on Ni-NTA agarose resin via this strategy has no detrimental effect on enzyme activity or stability. The lyoprotectant properties of Ni-NTA were also investigated. In Chapter 5, we designed and characterized a mutant of CYP3A4 that retains its enzymatic activity upon modification with a variety of fluorescent maleimide dyes via a single cysteine residue on its surface, namely C64. We also show that the activity of this mutant is preserved upon immobilization onto solid supports via this same cysteine residue. Finally, results of a preliminary feasibility study towards applying this immobilization strategy to eventual single-molecule fluorescence microscopy studies are presented.
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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 | 0.000 |
| 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) | 0.000 | 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".