Investigations of Self-Sufficient P450cam Monooxygenases for Activity and Enantioselectivity

  • Anja Eichler

Student thesis: Phd


Catalytic, selective C-H bond activation for the oxidative hydroxylation RH → ROH of simple or complex compounds is of significant interest in synthetic organic chemistry. One of the major classes of enzymes used for C-H bond activation are cytochrome P450 monooxygenases (EC 1.14.X.X), which can promote chemo-, regio- and stereoselective oxidations under mild reaction conditions. For the current study, catalytically self-sufficient forms of biocatalyst P450cam-RhFRed were investigated. These self-sufficient P450 systems were previously created by fusing the reductase domain of P450 RhF (CYP116B2, RhFRed from Rhodococcus sp.) with the catalytic domain of P450cam (CYP101A1, Pseudomonas putida), thus mimicking the natural fusion of P450 RhF. The generation of 93 P450cam-RhFRed variants has expanded the synthetic toolbox to serve as a basis for exploring the substrate scope towards ethylbenzenes, substituted alkylbenzenes, 4-ethylphenol and (+)-pleuromutilin. To select for active mutants from this library of 93, high throughput screening methods were developed. A pooling approach was applied in order to express P450s and analyse them against a panel of non-natural substrates, such as ethylbenzene, 4-ethylphenol and (+)-pleuromutilin in whole cell biotransformation reactions. The concentration of P450 enzymes was determined using CO difference spectroscopy in whole cells. The assay was significantly improved both in terms of speed and safety by using carbon monoxide releasing molecules as a source of CO rather than the gas CO itself. These screening studies served as starting point to identify P450cam-RhFRed mutants for specific reactions. In particular, a systematic investigation of this library showed mutants that generated chiral benzyl alcohols with good enantioselectivities.To interpret these results on a structural basis, molecular dynamics simulations were used to estimate enantioselectivity of selected mutants for the regio-isomers of methylated ethylbenzene derivatives. The results from the molecular dynamics simulations were broadly consistent with experimentally determined data and identified the importance of conformational changes and flexibility of mutant-substrate complexes to enforce enantioselectivity.
Date of Award31 Dec 2016
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorSabine Flitsch (Supervisor) & Simon Webb (Supervisor)


  • C-H activation
  • P450 enzymes
  • Selectivity

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