The exact chemical identity of reactive intermediates in O2-dependent uric acid biodegradation

Differently from the majority of other animals humans cope with large quantities of uric acid in their bodies. This is because during evolution we have progressively silenced a gene responsible for the production of an enzyme called urate oxidase (UOX). This enzyme is able to break down uric acid into more soluble compounds. The reasons for human adaptation to high uric acid levels are not entirely clear and, interestingly, mice without a functional UOX enzyme die shortly after birth. Under certain pathological conditions that cause a further increase in uric acid UOX is administered to patients to help restore normal levels. Crystalline uric acid deposits are also the hallmark of gout disease.

UOX requires molecular oxygen (O2) to perform its task of breaking down uric acid. O2 is a very interesting molecule as in its normal "resting" state (the form present in the air) does not want to react with the vast majority of organic molecules for reasons related to its electronic structure. Oxygen needs activation to react. A major problem, however, is that once "activated" oxygen can react indiscriminately with many biological molecules with detrimental consequences. For example, reactive oxygen species (ROS) are damaging forms of "active oxygen" that play an important role in aging. Therefore, besides the generation of "active oxygen", another challenge in oxygen biochemistry, is its control. In this work we will investigate how UOX uses O2 to break down uric acid. Interestingly, UOX belongs to a small group of enzymes that can bring oxygen into reacting with their organic substrates and steer the reaction towards the desired products with limited chemical tools at its disposal. In fact, as oxygen activation is not an easy task, the vast majority of enzymes rely on special additional components like metal and/or organic co-factors to form "active oxygen". UOX does not require these additional helpers and therefore understanding how it works is particularly intriguing.

Using a technique called X-ray crystallography which allows to 'see' at very high resolution the 3D structure of molecules as small as urate oxidase (10,000 times smaller that the thickness of a human's hair) we have been able to visualise snapshots of the enzyme along the process of uric acid degradation (reaction intermediates) including also the state in which O2 is trapped above the substrate. These snapshots led us to formulate some hypotheses on how urate oxidase works. We are now in an excellent position to study the most elusive and critically important properties of UOX chemistry. For this we will use a technique called neutron crystallography that can detect atoms (hydrogens) that cannot be typically observed even by X-ray crystallography. By combining neutron crystallography, X-ray crystallography, modern spectroscopic techniques and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible we will understand general rules of O2 biochemistry in the context of UOX function. This integrated approach will allow a deeper understanding not only of UOX but also of oxygen, an essential component of life on Earth.

The unambiguous detection of reaction intermediates is one of the most challenging tasks in mechanistic enzymology. In particular, transient organic peroxides are likely formed in many enzymatic reactions employing O2, yet, direct structural evidence for them is minimal, particularly, in the absence of metal centres. Using atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations we have recently shown that the archetypical cofactor-independent urate oxidase (UOX) catalyses uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Additionally, we demonstrated that very low X-ray doses break specifically the intermediate C5-OO(H) bond at 100 K, thus releasing O2 in situ. The latter is trapped above the substrate radical generated in the radiolytic process. Furthermore, the dose-dependent rate of bond rupture was followed by combined crystallographic and Raman analysis indicating that ionising radiation kick-starts both peroxide decomposition and its regeneration. We hypothesised that peroxidation occurs by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site. This radical-radical recombination stop is believed to be directly relevant to the catalytic process. Building on this mechanistic work we are now in an excellent position to extend our investigation to interrogate other challenging aspects of the UOX reaction mechanism with particular emphasis on the detailed protonation state of O2-dependent catalytic steps. Filling this gap in knowledge is key to understand how substrate-O2 activation is achieved. The UOX system is uniquely suited to an integrated experimental and theoretical approach in which we will employ neutron (cryo)crystallography, atomic-resolution X-ray crystallography, in crystallo spectroscopies and advanced QM/MM methods to understand oxygen-dependent catalysis in a metal-independent framework.

The team is committed to ensuring that every aspect of this work will achieve impact. The following three main objectives will be pursued.

1. Cross-discipline research. The training the PDRA (Dr Soi Bui) will gain from this research will equip him with a rare and sought-after skill-set as well as a comprehensive overview of a cross-discipline study. In particular, he will be able to expand his X-ray crystallographic and spectroscopic training by incorporating hands-on training in neutron crystallography performed at a world-leading centre in neutron science (ILL, Grenoble). The fact that of the 83 neutron structures present in the Protein Data Bank (PDB), more than half (49/83) were deposited since 2010 suggests that this technique will likely see an expansion in the future thanks to a number of technical advances that make experiments simpler than in the past. Experience gained during this project will enable him to make a valuable and practical contribution to the continued growth of cutting-edge molecular enzymology activities in the UK. Beyond these specific scientific skills, the coordination of this multi-faceted research project will give him invaluable experience in a number of areas applicable to much of the employment sector. These include: people and time management; budgeting; responsible and thorough communication of results and ideas; coordination of personnel with a wide variety of expertise and interests in achieving a common aim. This project offers also an ideal opportunity for further push collaborative work between the groups at King's College London and between King's and the ILL.

2. Communication and engagement. The College puts an enormous effort and provides excellent support for scientists to communicate their work to the public. These range from public lectures to direct contact with local schools. We will fully engage with this infrastructure to ensure that our ideas are explained to the widest audience possible. For example, King's has an active role in London Higher - Supporting Higher Education in London Programme of widening participation events. Taster days, master classes, homework clubs, workshops, and study skills sessions focus on students who have had no previous family experience of Higher Education. Evaluation forms collected from past events have been extremely positive, with many learners citing the engaging and inspirational input as having raised their aspirations and given them an entirely new perspective on University life and study. As part of King's outreach program, the Randall Division and the more specifically the Steiner lab (in the summer break), also welcome secondary school students from various Colleges to experience life in an active research laboratory.

3. PDRA transferable training. The PDRA will have access to the full training and development infrastructure that is available at King's College. As well as a first class scientific training in the department and contacts with world class scientists visiting the Randall Division, access is available to a plethora of courses available through the King's Learning Institute which will all them to develop non-academic skills ranging from IT to leadership. This will enhance his skills set and make him very competitive for the next stage of his career in either academia or the industrial/biotechnology sector. The PI is committed to supporting his career development in whatever direction he chooses to go.

Specific milestones are provided in the Pathway to Impact document.