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Engineering and Characterization of Cytochrome P450 Enzymes for Nitrogen-Atom Transfer Reactions


Farwell, Christopher C. (2015) Engineering and Characterization of Cytochrome P450 Enzymes for Nitrogen-Atom Transfer Reactions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9765C71.


The creation of novel enzyme activity is a great challenge to protein engineers, but nature has done so repeatedly throughout the process of natural selection. I begin by outlining the multitude of distinct reactions catalyzed by a single enzyme class, cytochrome P450 monooxygenases. I discuss the ability of cytochrome P450 to generate reactive intermediates capable of diverse reactivity, suggesting this enzyme can also be used to generate novel reactive intermediates in the form of metal-carbenoid and nitrenoid species. I then show that cytochrome P450 from Bacillus megaterium (P450BM3) and its isolated cofactor can catalyze metal-nitrenoid transfer in the form of intramolecular C–H bond amination. Mutations to the protein sequence can enhance the reactivity and selectivity of this transformation significantly beyond that of the free cofactor. Next, I demonstrate an intermolecular nitrene transfer reaction catalyzed by P450BM3 in the form of sulfide imidation. Understanding that sulfur heteroatoms are strong nucleophiles, I show that increasing the sulfide nucleophilicity through substituents on the aryl sulfide ring can dramatically increase reaction productivity. To explore engineering nitrenoid transfer in P450BM3, active site mutagenesis is employed to tune the regioselectivity intramolecular C–H amination catalysts. The solution of the crystal structure of a highly selective variant demonstrates that hydrophobic residues in the active site strongly modulate reactivity and regioselectivity. Finally, I use a similar strategy to develop P450-based catalysts for intermolecular olefin aziridination, demonstrating that active site mutagenesis can greatly enhance this nitrene transfer reaction. The resulting variant can catalyze intermolecular aziridination with more than 1000 total turnovers and enantioselectivity of up to 99% ee.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Enzyme catalysis, metal-nitrenoid, directed evolution, protein engineering, asymmetric C-H amination, aziridination, sulfimidation
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Tirrell, David A. (chair)
  • Miller, Thomas F.
  • Gray, Harry B.
  • Arnold, Frances Hamilton
Defense Date:27 January 2015
Funding AgencyGrant Number
National Science Foundation Graduate Research FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:01302015-155404718
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for ch. 1 adapted for ch. 2 adapted for ch. 3 adapted for ch. 4
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8764
Deposited By: Christopher Farwell
Deposited On:26 Mar 2015 21:45
Last Modified:08 Nov 2023 00:11

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