Structural, Mechanistic and Functional Studies on Protein Hydroxylases
Lead Research Organisation:
University of Oxford
Department Name: Oxford Chemistry
Abstract
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.
Technical Summary
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.
Planned Impact
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.
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.
Organisations
Publications
Abboud MI
(2018)
2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2.
in Chemical communications (Cambridge, England)
Aik W
(2014)
Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation.
in Nucleic acids research
Brewitz L
(2020)
Kinetic parameters of human aspartate/asparagine-ß-hydroxylase suggest that it has a possible function in oxygen sensing.
in The Journal of biological chemistry
Brewitz L
(2020)
Aspartate/asparagine-ß-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors.
in Scientific reports
Brewitz L
(2020)
Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-ß-hydroxylase.
in Bioorganic & medicinal chemistry
Böttger A
(2015)
The oxygenase Jmjd6--a case study in conflicting assignments.
in The Biochemical journal
Chan M
(2016)
Pharmacological targeting of the HIF hydroxylases - A new field in medicine development
in Molecular Aspects of Medicine
Choi H
(2020)
A human protein hydroxylase that accepts D-residues.
in Communications chemistry
Chowdhury R
(2014)
Ribosomal oxygenases are structurally conserved from prokaryotes to humans.
in Nature
Domene C
(2020)
Mechanism of Molecular Oxygen Diffusion in a Hypoxia-Sensing Prolyl Hydroxylase Using Multiscale Simulation.
in Journal of the American Chemical Society
Figg WD
(2021)
Structural Basis of Prolyl Hydroxylase Domain Inhibition by Molidustat.
in ChemMedChem
Grover A
(2016)
Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.
in Nature communications
Hamed RB
(2016)
Use of Methylmalonyl-CoA Epimerase in Enhancing Crotonase Stereoselectivity.
in Chembiochem : a European journal of chemical biology
Heim A
(2014)
Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine-serine-rich (RS) domains of SR- and SR-like proteins.
in Nucleic acids research
Hopkinson RJ
(2018)
Human histone demethylase KDM6B can catalyse sequential oxidations.
in Chemical communications (Cambridge, England)
Hsu KF
(2021)
Hypoxia and hypoxia mimetics differentially modulate histone post-translational modifications.
in Epigenetics
Islam MS
(2022)
Inhibition of JMJD6 by 2-Oxoglutarate Mimics.
in ChemMedChem
Islam MS
(2018)
2-Oxoglutarate-Dependent Oxygenases.
in Annual review of biochemistry
Langley GW
(2016)
Analysis of JmjC Demethylase-Catalyzed Demethylation Using Geometrically-Constrained Lysine Analogues.
in ACS chemical biology
Langley GW
(2019)
Profiling interactions of vaborbactam with metallo-ß-lactamases.
in Bioorganic & medicinal chemistry letters
Lesniak RK
(2019)
19F NMR studies on ?-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode.
in Chemical communications (Cambridge, England)
Lohans CT
(2019)
Non-Hydrolytic ß-Lactam Antibiotic Fragmentation by l,d-Transpeptidases and Serine ß-Lactamase Cysteine Variants.
in Angewandte Chemie (International ed. in English)
Lu M
(2014)
A Code for RanGDP Binding in Ankyrin Repeats Defines a Nuclear Import Pathway
in Cell
Markolovic S
(2018)
The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.
in Nature chemical biology
Markolovic S
(2016)
Structure-function relationships of human JmjC oxygenases-demethylases versus hydroxylases.
in Current opinion in structural biology
Markolovic S
(2015)
Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases.
in The Journal of biological chemistry
Nowak RP
(2021)
First-in-Class Inhibitors of the Ribosomal Oxygenase MINA53.
in Journal of medicinal chemistry
Sekirnik R
(2018)
YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase.
in Extremophiles : life under extreme conditions
Smart TJ
(2020)
Studies on the selectivity of proline hydroxylases reveal new substrates including bicycles.
in Bioorganic chemistry
Thinnes C
(2019)
Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding
in Chemistry - A European Journal
Wilkins SE
(2018)
JMJD5 is a human arginyl C-3 hydroxylase.
in Nature communications
Wilkins SE
(2016)
Targeting Protein-Protein Interactions in the HIF System.
in ChemMedChem
Williams ST
(2014)
Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates.
in Epigenetics
Xu C
(2014)
Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation.
in The Journal of biological chemistry
Yeh TL
(2017)
Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials.
in Chemical science
Description | 1st RSC Prize lecture, Reading |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | "Remarkable Enzymes - from Bacteria to Humans" |
Year(s) Of Engagement Activity | 2019 |
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Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | This course aims to disseminate the cutting-edge research about metals in biology and medicine to Hong Kong. The exploration of metals in biology and medicine is a fast-growing research area. Over the past decades, copper, iron and zinc have attracted enormous attentions due to the involvement of metals in aging and neuroscience. These metals can be detected and monitored in vivo by multi-techniques and multi-omics approaches, which further our understanding in metal homeostasis and metals in pathophysiological processes. This workshop aims to share some insightful views and ideas in molecular imaging and biosensing, metals in medicine, antibiotics and drug resistance |
Year(s) Of Engagement Activity | 2021 |
URL | https://chemistry.hku.hk/events/seminars_conferences_detail/175/ |
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Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
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Year(s) Of Engagement Activity | 2019 |
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Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
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Year(s) Of Engagement Activity | 2019 |
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Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Talk at Informa Life Sciences' Enzymes Event, London on "Protein Hydroxylation by Oxygenases" |
Year(s) Of Engagement Activity | 2015 |
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Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Study participants or study members |
Results and Impact | "The chemistry of prolyl-hydroxylases and their inhibition" |
Year(s) Of Engagement Activity | 2019 |
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Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | Talk at Proteomics Meeting COST, Oxford on "Proteomics for Functional Assignment of Oxygenases (is Hard Work)" |
Year(s) Of Engagement Activity | 2015 |
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Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | This event brought around 100 researchers together in-person and virtually. Attendees were from many of the Medical Sciences Division departments, some Mathematical, Physical and Life Sciences Division departments, MRC Harwell, and the Novo Nordisk Research Centre Oxford. In addition, we were joined by guests from Novo Nordisk, Denmark. The symposium was an informative and enjoyable day of talks, posters and networking, and a brilliant opportunity for all the fellows, including past fellows, and their mentors to reconnect. |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.rdm.ox.ac.uk/work-with-us/novo-nordisk-oxford-fellowships/symposium |
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Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
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Year(s) Of Engagement Activity | 2021 |
URL | https://www.southampton.ac.uk/news/events/distinguished-lecture-series.page |
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Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | "Adventures in Functional Assignment and Inhibition of Metallo-Enzymes" |
Year(s) Of Engagement Activity | 2019 |
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Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Talk at Proxomics Meeting, Imperial College London on "Assigning New Roles for Protein Hydroxylation" |
Year(s) Of Engagement Activity | 2015 |
Description | Uppsala University, Sweden |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Study participants or study members |
Results and Impact | 'From penicillins to epigenetics' |
Year(s) Of Engagement Activity | 2019 |
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Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | "From antibiotics to epigenetics - adventures at the interface of chemistry and biology" Visiting period 26-29 August and 15-18 September |
Year(s) Of Engagement Activity | 2019 |