Cytochrome P-450 monoxygenases catalyse the insertion of molecular oxygen into unactivated carbon-hydrogen bonds. This reaction is difficult to reproduce synthetically, therefore cytochrome P-450 enzymes are of great interest as potential biocatalysts. Of all the cytochrome P-450 systems investigated to-date cytochrome P-450cam form <i>Pseudomonas putida </i>is probably the best characterized. The use of cytochrome P-450cm, as a biocatalyst, is hampered by the narrow substrate range. This was overcome by use of the cytochrome P-450cam Y96A mutant. To identify the substrate range of the enzyme a model of the cytochrome P-450cam binding site was generated and potential substrates were modelled. Discrepancies between predicted and observed results led to a more rigorous study of the mutant binding site. Conditions were developed and the cytochrome P-450cam mutant enzyme was crystallized in the presence of a number of substrates. Six different substrate bound structures were then solved by X-ray crystallography. With a crystal structure of the binding site it was then possible to perform more rigorous molecular modelling and this was achieved with modelling program DOCK. Biotransformations and binding studies were performed with a range of substrates to test the validity of the modelling and to characterize the substrate specificity of the cytochrome P-450cam Y96A mutant.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:662348 |
| Date | January 2000 |
| Creators | Staines, Adam G. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14476 |
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