Structural, Mechanistic and Functional Studies on Oxgenases

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).

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.

We are committed to ensuring that the proposed publicly-funded work on oxygenases achieves substantial impact outside of academia / research. Two fields in which this will occur in a project specific manner are antimicrobial resistance (AMR) and clinical work relating to the manipulation of the natural hypoxic response.

AMR related impacts: The work will provide insight into the mechanisms by which beta-lactams, which are the most important antibiotics, are biosynthesized. Work on breakthrough new drugs in the field has substantially been limited by (bio)synthetic challenges, meaning much research has focused on old synthetically facile classes. Our proposed work on isopenicillin N synthase (IPNS)/related enzymes will enable production of new types of beta-lactamase/penicillin binding protein inhibitor and routes to useful antibiotics by engineering. In addition to normal outputs (primary publications, reviews patents) to optimise the impact of our work in the AMR field we will give at least 3 lectures a year to industrial, clinical, academic and charitable initiatives on AMR.

Hypoxia / oxygenase related impacts. Our proposed oxygenase work and on how oxygen interacts with them is of interest to the large community working on life forms in which oxygenases post-translational hydroxylation occurs, from prokaryotes to animals. Human 2OG oxygenases are linked to ageing, healthy development and diseases. HIF prolyl hydroxylase inhibitors are recently approved for anaemia treatment and others in are in late stage clinical development, but all are blunt active site blockers which, despite efficacy, are imperfect in terms of selectivity over other 2OG oxygenases, an important issue in anaemia treatment, where patients may be taking drugs for decades. Our work suggests that it will be possible to develop better compounds with allosteric modes of action, with ones that modulate the kinetics of oxygen binding of particular interest. In addition to the normal channels (below), we will contact all companies known to be working in the field on an annual basis to update them with key results with the aim of helping them make better /safer medicines and to help them interpret results of ongoing clinical trials.

Other project specific deliverables: (i) 2 papers on time-resolved IPNS studies on IPNS; (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 a human HIF prolyl hydroxylase; (iv) 3 papers including time-resolved studies on other 2OG oxygenases, with one focusing on oxygen transport; (v) A comprehensive review on the roles of oxygenases in the hypoxic response (year 2); (vi) A short review on the use of XFEL for mechanistic enzymology of metallo-proteins; (vii)Depositing >40 structures to the PDB during the course of the work; (viii) There is a very high chance the work will lead to IP - we have a good track record in this regard and relationship the technology transfer arm of Oxford University; (ix) We will use communication platforms to make our findings accessible to many potential stakeholders. We will select conferences based on expected stakeholder participation. We will engage with local clinical communities by maintaining and strengthening links with bodies such as Antibiotic Research UK. Engagement activities will include public (e.g. Pint of Science) / schools talks, open day participation and innovative events such as exhibiting materials related to antimicrobial discovery; (x) We will engage the BBSRC/other stakeholders in communications activities. BBSRC funding will be acknowledged in communications with both fellow researchers and the general public and that BBSRC branding is used. We will engage with BBSRC communications by using social media to share research; (xi) Research Fish will be used to record communication/engagement activities.