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(BBSRC DTP CASE) Exploitation of P450 BM3 variants for production of valuable drug metabolites


Project Description

The US Food and Drug Administration (FDA) have recommended that human drug metabolites (as well as their parent drugs) should be subject to testing for safety/toxicity. Most such primary metabolites are produced in vivo by human cytochrome P450 enzymes. However, the metabolites are difficult to obtain from human cells and their chemical synthesis is expensive and laborious. Ideally, a robust, soluble and catalytically efficient microbial P450 enzyme could be used for this purpose, subject to its being engineered to bind drug substrates and to transform them into bona fide drug metabolites. Such an enzyme is P450 BM3, a natural fusion of a P450 to its reductase partner, and which has the highest activity reported for any P450 enzyme with its natural fatty acid substrates. We have engineered P450 BM3 to (i) alter its conformational flexibility, and (ii) introduce disruptive proline mutations in the P450 core. This P450 re- engineering results in (i) variants that recognize and oxidize multiple novel compounds that are not substrates for wild-type BM3, and (ii) other mutants that alter the binding mode for fatty acids in the active site, resulting in oxidation of lipids at various different positions. Novel substrates for BM3 mutants include pharmaceuticals and other important molecules such as proton pump inhibitors (e.g. omeprazole), steroids, nystatin, artemisinin and paclitaxel. This PhD project will exploit these findings and the appointee will analyse the products formed from the diverse compounds bound to BM3 mutants in order to identify human drug metabolites. This will be done with a view to their scale-up and ultimately for their commercialization and applications including drug safety testing. Further protein engineering will also be done on the proline-mutated BM3 variants to produce a range of different fatty acids oxidized at different positions. The successful candidate will receive extensive training at the MIB in Manchester (including protein expression/engineering, P450 crystallization and structural determination, compound screening and identification of new substrates for BM3 variants, and analytical studies to identify metabolites formed from key substrates). At the industrial partner Cypex Ltd, the appointee will work on the production of metabolites using in vitro methods, in addition to using fermentation of BM3 mutant expression cells permeabilised to facilitate drug entry. The project will provide the student with broad training in enzymology, structural biology, microbiology and analytical techniques, and will equip the appointee with important skills relevant for a career in industry or academia.

http://www.mib.ac.uk
https://www.research.manchester.ac.uk/portal/Sam.Hay.html
https://www.research.manchester.ac.uk/portal/david.leys.html
https://www.research.manchester.ac.uk/portal/andrew.munro.html

Entry Requirements:
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Partnership. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP website View Website

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

1) Munro AW et al. (2002), P450 BM3: the very model of a modern flavocytochrome. Trends Biochem. Sci. 27, 250-257.
2) Girvan HM and Munro AW (2016), Applications of cytochrome P450 enzymes in biotechnology and synthetic biology. Curr. Opin. Chem. Biol. 31, 136-145.
3) Richards L et al. (2019), Production of metabolites of the anti-cancer drug noscapine using a P450 BM3 mutant library. Biotechnol. Rep. 24:e00372.
4) Butler CF et al. (2013), Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias. J. Biol. Chem. 288, 25387-25399.
5) Butler CF et al. (2014), Human P450-like oxidation of diverse proton pump inhibitor drugs by ‘gatekeeper’ mutants of flavocytochrome P450 BM3. Biochem. J. 460, 247-259.

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