Cytochromes P450 (P450s) are a superfamily of heme-thiolate monooxygenases. They catalyse a wide variety of reactions on a vast number of substrates and are of particular interest for biocatalyst development due to their ability to oxidise non-activated C-H bonds. Fusion of a P450 to a suitable redox partner protein produces a catalytically self-sufficient enzyme and removes the need to produce electron transfer proteins separately. The well-studied bacterial protein P450cam (Pseudomonas putida) has been fused to the reductase (RhFRed) from the natural fusion protein P450-RhF (Rhodococcus sp.). The P450cam-RhFRed system catalyses the oxidation of camphor and several non-natural substrates and served as the basis for P450cam re-engineering in this current project, with the aim of expanding the substrate scope towards a more mammalian-like activity. The P450cam active site was partitioned into seven paired amino acids and each pair randomised in turn to generate seven sub-libraries of P450cam variants. These were screened for activity using a specially developed colony screen for detection of the blue pigment indigo. In total 94 new variants were identified and then pooled for secondary screening on a number of new substrates, identifying potentially novel activities within the ‘indigo positive’ population. In a separate ‘chimeragenesis’ approach substrate recognition sites (SRSs) within P450cam were targeted for exchange with equivalent portions from a number of human P450s. The B’ helix and F-G loop regions from CYPs 1A2, 2C8, 2D6 and 3A4 were grafted onto the P450cam structure and several of the B’ helix swaps were produced as soluble proteins. The P450cam-2C8-B’-RhFRed chimera gave a Soret peak at 420 nm in the Fe(II)-CO state although an additional substitution next to the proximal cysteine appeared to restore a P450-like state. SRS-exchange therefore offered some insight into structural modularity in P450s, providing a basis for further biocatalyst development.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626937 |
| Date | January 2014 |
| Creators | Kelly, Paul |
| Contributors | Flitsch, Sabine; Turner, Nicholas |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/engineering-cytochrome-p450reductase-fusion-enzymes-for-biocatalysis(c4b3aa48-1c73-4980-b480-e3a881267ee5).html |
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