Structural, Mechanistic and Functional Studies on Oxgenases

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Following substantially from prior BBSRC work Fe(II) and 2-oxogluturate (2OG) dependent oxygenases and related enzymes have emerged as important catalysts across much of biology. In microorganisms they catalyse key steps in the biosynthesis of antibacterials, including in production of the beta-lactams such as penicillins. In humans 2OG oxygenases have key roles in collagen biosynthesis, nucleic acid repair/modification, histone demethylation, lipid metabolism and, the physiologically important animal hypoxic response. Building on extensive BBSRC work we aim to carry out detailed mechanistic studies, including time resolved crystallographic, NMR and MS work, on the mechanisms of 2OG oxygenases and the related enzyme isopenicillin N synthase (IPNS). The work will encompass time resolved crystallography employing X-ray free electron laser (XFEL) and conventional crystallography methodologies, coupled with solution studies (notably by protein observed NMR and MS), and modelling. The results will inform on the precise mechanisms by which dioxygen is transported from the surface of the protein to the active site and on dynamic changes during catalysis. Extensive preliminary studies, including using XFEL, with IPNS reveal the viability of the approach and its ability to reveal unexpected conformational changes during catalysis. The results will provide basic insight into how enzymes of major societal importance work. The utility of the mechanistic work will be demonstrated in functional assignment work on human 2OG oxygenases, will be of use in making improved inhibitors for treatment of diseases such as anaemia, and in engineering IPNS to produce beta-lactam products with improved antibacterial activity and resistance to beta-lactamases.

The enzymes we will study are of interest from antimicrobial resistance and human physiology (hypoxia, epigenetics) perspectives; the results have potential to attract substantial public and media interest (as the case with our previous BBSRC work). The impact of the work will extend from academia through to the public (health care) and private (industrial/pharmaceutical) sectors to policy influencers and the general public. We will aim to publish at least 6 papers in high quality journals (e.g. Nature, Cell, PNAS) during the work, i.e. one for each PDRA in each year.
Project specific deliverables include:
(i) Two papers on time-resolved mechanistic studies on isopenicillin N synthase.
(ii) A study comparing radiation damage on metallo-enzymes comparing results with different data collection methods.
(iii) A major study on time resolved studies on the human hypoxia inducible factor prolyl hydroxylase 2.
(iv) Three papers including time-resolved mechanistic studies on other 2-oxogulturate dependent oxygenases, including at least one focusing on dioxygen transport within the enzymes.
(v) A comprehensive review on the roles of oxygenases in the hypoxic response.
(vi) A short review on the use of XFEL for mechanistic enzymology of metallo-proteins
(vii) We will deposit >40 structures to the PDB during the course of the work.
(viii) Aside from the academic inputs resulting from publications, there is a good chance that the work will lead to intellectual property of commercial value - we have a good track record in the regard and relationship with OUI, the technology transfer arm of Oxford University.
(ix) The work will also benefit the large number of medical chemists working on AMR and the hypoxic response. Although some inhibitors of the HIF prolyl hydroxylases have been approved for clinical use (for anemia treatment), these are 'blunt' active site blockers. Our mechanistic insights will help enable development of compounds that modulate rather than block activity (e.g. by allosteric binding that alters kinetics).
(x) We will use a range of communication platforms to make our findings accessible to as many potential stakeholders as possible. Aside from peer-reviewed publications we will select conferences based on expected stakeholder participation (clinicians, industrial researchers) outside our immediate academic community, e.g. British Society for Antimicrobial Chemotherapy AMR mechanisms workshop or the American Society for Microbiology Microbe and Gordon/Keystone conferences on hypoxia/epigenetics. We will engage with local clinical communities by maintaining and strengthening links with bodies such as Antibiotic Research UK (CJS is a scientific advisor) and Oxford National Institute for Health Research Biomedical Research Centres. Public engagement activities will include public (e.g. Pint of Science) and schools talks, open day participation and innovative events such as exhibiting materials related to antimicrobial discovery, as in the "Back from the Dead" museum exhibition to which Co-I CJS contributed.
(xi) We will engage the BBSRC (and other stakeholders i.e. Diamond Light Source) in communications activities, e.g. by notifying Press Offices when significant publications are accepted/impacts made. BBSRC funding will be acknowledged in communications with both fellow researchers and the general public and that BBSRC branding is evident in slides and posters and on websites and other visual media. We will engage with BBSRC communications by using social media (e.g. Twitter) to share BBSRC research following University and BBSRC guidelines.
(xii) We will highlight roles of the early career researchers of raising awareness of the opportunities and rewards of research careers. We will use Research Fish to record communication and engagement activities. We will be conscious of diversity issues at all stages in the work, from appointments to promoting the careers of early stage researchers.