Award details

Structural, Mechanistic and Functional Studies on Oxgenases

Reference	BB/V001248/1
Principal Investigator / Supervisor	Dr Allen Orville
Co-Investigators / Co-Supervisors
Institution	Diamond Light Source
Department	Science Division
Funding type	Research
Value (£)	22,272
Status	Current
Type	Research Grant
Start date	01/06/2021
End date	31/05/2024
Duration	36 months

Abstract

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.

Summary

Proteins are polymers that are crucial to all aspects of life and which are biologically produced by polymerisation of monomeric amino acid precursors. In the early 20th century evidence was reported (by Henry Dakin) that proteins can react with atmospheric oxygen. Later it was found that penicillins are made from a tripeptide (three linked amino acids) by reaction with oxygen. Much more recently it was found that proteins that regulate how humans and other animals respond to limiting oxygen availability (hypoxia) are modified by direct reactions with oxygen, in a manner that decreases their activity. The reaction of these hypoxia inducible factors (HIFs) with oxygen signals for their degradation, so turning on the hypoxic response. The hypoxic response is of massive importance in human biology. If we go to high altitude we make more red blood cells to compensate for the reduced oxygen availability. Red blood cell production is stimulated by increases in the level of a hormone called erythropoietin (EPO), which in turn is increased by the HIFs. EPO is a really important medicine for the treatment of anaemia, but is an expensive protein to make and is not suitable for use by all anaemia sufferers. The hypoxic response is also important in cancer and the development of healthy animal physiology. If HIF degradation can be blocked by a drug EPO levels will increase and in turn red blood cell levels will increase, i.e. there is a new treatment for anaemia. PHD inhibitors have been developed but these are rather blunt instruments. Remarkably, the same family of oxygenases, i.e. enzymes using oxygen for catalysis, includes members that catalyse formation of penicillins from a tripeptide precursor (in effect a tiny protein) and enzymes that are the key regulators of the HIF mediated human hypoxic response - the PHDs. Previous BBSRC work has enabled the characterisation of these two types of oxygenases and shown related enzymes have roles in many other aspects of biology,including lipid metabolism and in the rapidly emerging field of epigenetics. Exactly how the oxygenase proteins interact with oxygen is not, however, well understood. In our new proposed BBSRC work we aim to elucidate the details of this process. In doing so we aim to inform on how dissolved gases interact with proteins in general, something of fundamental interest in biology, including from an evolutionary perspective, but on which there has been relatively little research. The results of our work will inform on how to make new antibiotics and make improved drugs for the treatment of anaemia and other hypoxic related diseases. The work will also enable the UK to remain at the forefront of basic science in research on oxygenases, a field of intense interest (as recognised by the 2019 Nobel prize for studies on the mechanisms of the human hypoxic response to which UK research contributed).

Impact Summary

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.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/V001892/1 Structural, Mechanistic and Functional Studies on Oxgenases

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search