Structural, Mechanistic and Functional Studies on Protein Hydroxylases

Proteins are polymers that are crucial to all aspects of life. Proteins are biologically produced polymers that are synthesised by polymerisation of monomeric amino acids. The template for the polymerisation process is messenger ribonucleic acid (mRNA), which in turn is encoded for by DNA, which is used for relatively long-term storage of information in the cells of all living organisms. However, once they have been synthesised, proteins can be further modified in processes that are often crucial for their physiological function. One such process is reaction with atmospheric oxygen, a small and high diffusible molecule. We are interested in defining how and why proteins react with oxygen from the atmosphere. In pioneering work it was found that atmospheric oxygen reacts with collagen, a material which helps cells to stick together in animals, in a reaction catalysed by oxygenases. Oxygenases are types of enzymes (or biological catalysts), that incorporate atmospheric oxygen into their reaction products. Many oxygenases use a metal, such as iron, to help capture oxygen. Subsequent to the discovery of its role in collagen biosynthesis, it was found that oxygenases play key roles in the production of antibiotics, such as the penicillins. More recently, we have found that oxygenases also catalyse the hydroxylation of proteins. Some of the protein targets of oxygenases are important from biological and medicinal perspectives. A breakthrough was the discovery that the physiological mechanism by which cells in animals respond to limiting oxygen is actually regulated by oxygenase catalysed hydroxylation of proteins, involved in regulating the conversion of DNA to mRNA. Following this discovery we, and others, have found other protein-hydroxylases, acting on a range of protein-residues. We are now in an exceptionally good position to work out how these enzymes work, including developing an understanding of how they bind their protein substrates. We will use crystallographic and other techniques, that will provide detailed information on how the enzymes work as machines. The structural and mechanistic studies will lay the groundwork in order to exploit the basic science to artificially alter the activity of the oxygenases, using them for the production of high-value modified proteins, and to provide knowledge that will be useful for the pharmaceutical industry in targeting them for diseases. Overall the work will enable the United Kingdom to remain at the forefront of basic science research on oxygenases and the exploitation of this research for the development of new medicines and catalysts for high value chemical production.

Following substantially from prior BBSRC work, protein-hydroxylation, as catalysed by 2-oxoglutarate (2OG) dependent oxygenases, has emerged as a common post-translational modification in eukaryotic, including human, cells. However, there are no reported structures for 2OG oxygenases in complex with intact protein substrates, and how the kinetic properties of the protein hydroxylases are related to their physiological roles in hypoxic sensing, transcription translation, and RNA splicing, is unclear. Building upon very extensive preliminary BBSRC funded work we aim to carry out definitive structural and kinetic studies on 2OG oxygenases catalysing hydroxylation of arginine-, lysine-, histidine- and other protein- residues. We will carry out combined structural, functional and mechanistic studies on human protein hydroxylases of biological importance. The structural studies will employ crystallography enzyme-protein complexes, and other techniques including non-denaturing MS, ITC and SPR. The kinetic studies will employ stopped-flow and flow-quench analyses coupled to MS and NMR. A particular focus will be to define the oxygen dependence of the enzymes. The results with respect to isolated proteins will be correlated with in-cell studies employing quantitative MS-based methodologies. Building upon exciting preliminary results, showing that one human hydroxylase is extremely promiscuous, accepting substrate residues ranging from asparagine- and aspartate- to leucine-, we will scope the utility of 2OG dependent protein hydroxylases for the site specific modification of residues and sequences of choice. Our objective is to enable the development of a set of oxygenases for the residue- and sequence- selective hydroxylation of proteins of choice. The work is enabled by the very large set of reagents and capabilities for studies on 2OG oxygenases that we have assembled, and will enable the UK to maintain a leading position in studies on protein-hydroxylation.

We are committed to ensuring that our publicly-funded work achieves substantial impact which in terms of translating fundamental research will make impacts on the health of people.
We believe results of work on the proposed project will be of interest to a very wide area of researchers from a range of scientific communities, in part because it has implications for the post-translational modifications in organisms ranging from some prokaryotes to yeast. The work is also of interest to the structural biology community, particularly those working on redox proteins, because there are no reported structures for 2OG oxygenases in complex with intact proteins, or the chemical community because 2OG oxygenases catalyse reactions presently impossible for synthesis. It is also of interest to the UK pharmaceutical industry, which is important to our economy, hence it will enable medicinal chemistry with respect to our new targets for small-molecules. Finally it is also of interest to the industrial biotechnology industry because of its potential to enable the site selective modification of proteins; this is useful for fold stabilization and for enabling the introduction of other post-translational modifications. During the three year lifetime of the project we will report the results via a minimum of six publications in high impact journals, writing a comprehensive review on oxygenases patent applications (if appropriate), depositing a minimum of 15 new oxygenase / oxygenase complex structures in the protein data bank, release probe compounds and procedures and, if appropriate, spin-out company formation.