Catechol-O-methyltransferase (COMT): Resolving the mechanism of an archetypical methyl transferase with new experimental tools

The primary means of deactivation of catecholamine neutrotransmeters such as dopamine, levodopa and (nor)epinephrine occurs through the action of an enzyme called catechol-O-methyltransferase (COMT). Consequently, inhibition of COMT is a strategy for the treatment of a range of neurological disorders including Parkinson's disease, depression and schizophrenia. In addition to being a drug target, COMT has potential as a useful biocatalyst to create valuable chemicals such as vanillin, the primary component of the extract of the vanilla bean. Clearly a detailed understanding of how COMT works would be of value to a range of sectors. However, despite more than 60 years of study, there are still major disagreements as to fundamental aspects of the physical mechanism of the reactions catalysed by methyl transferases such as COMT.

The focus of this work is to study COMT using new experimental tools - NMR and optical spectroscopies, X-ray crystallography - alongside a tightly integrated computational chemistry programme, with a focus on the physical mechanism of catalysis by this enzyme. This work will capitalise on our recent work, which produced the first NMR backbone assignments of COMT, which is necessary to map NMR data onto new and/or existing X-ray crystal structure data. We will extend this approach to study how COMT binds a range of physiological substrates such as dopamine. By using our NMR experiments as a screening method, we expect to optimise sample conditions so we can solve the first X-ray crystal structures of COMT with bound catecholamines, allowing better description of neurotransmitter binding to COMT.

We will expand on our previous NMR work to perform more technically demanding high pressure and relaxation NMR experiments that directly probe protein flexibility and dynamics. Alongside optical fluorescence and vibrational experiments, these experiments will allow the identification of any specific dynamic features - amino acid backbone and/or side chain motions - that may contribute to catalysis. The significance of such behaviour will be investigated by altering amino acids of interesting (by site-directed mutagenesis) and by comparing the activity (enzyme turnover) of wild-type and mutant enzymes lacking amino acids of interest. This approach allows the direct assessment of dynamical features to COMT catalysis - a major area of controversy in the wider enzymology community.

A major feature of the proposal is the close integration of computational simulations with experimental work, which will be performed both in Manchester and by collaborators at the University of Arizona. Experiments are designed to provide data that can be directly used as inputs to computational calculations. Calculations and simulations will be directly tested/benchmarked against experimental data to allow successive improvements to computational parameters and to guide the design of subsequent experiments, e.g. in the identification of amino acids that may play a major role in catalysis. Together, this approach will allow new insight into how COMT - and by extension, related enzymes - works. In addition to resolving long-standing questions in the field, this work could lead to improvements in drugs designed to inhibit COMT and related enzymes, will provide foundational data that will aid in the rational (re)design of related methyl transfer enzymes for use in biocatalysis applications, and will provide new methodology to tackle similar mechanistic questions in a wide range of other enzymes.

While COMT is an established drug target, controversies remain as the physical origin of catalysis in this and related enzymes. COMT has long been a focus of the computational chemistry community, but a major issue is that until recently computational studies have been based on relatively few experimental data: KIE measurements of COMT and reference reactions, steady state inhibition assays and X-ray crystal structures of inhibitor complexes. We suggest that progress towards a consensus description of catalysis by COMT - and by extension other methyl transferases - requires new experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of poises along the reaction coordinate (e.g. reactant and transition states).

We have the unique expertise required to prepare COMT samples that are capable of giving good NMR resolution and that readily crystallise and diffract to high resolution (1.3 A). Having recently completed backbone assignments of two ternary complexes of COMT, we will extend this work to include additional ternary complexes containing physiologically relevant catecholamines and/or transition state-like analogues. We will also use new and existing assignment and structural data to gain a deeper understanding of how COMT catalyses methyl transfer via a range of new NMR experiments including high pressure and relaxation measurements aimed at probing the flexibility and dynamics of the enzyme. Specific features (amino acids) of interested will be targeted for site-direct mutagenesis, with their functional contributions quantified by comparison of enzyme kinetics of wild-type and mutant COMT.

To fully realize the potential of new and existing COMT data, we will use a range of computational chemistry approaches including MD simulations, DFT modelling, and in collaboration with the Schwartz group QM/MM calculations to model structural and functional aspects of COMT.

As this is fundamental research, early impact will likely be largely felt in related academic and industrial scientific communities. Impact activities will focus on knowledge exchange to both specialist and non-specialist audiences.

Beneficiaries:
Enzymes such as COMT are central to life systems. Our understanding of catalysis underpins the exploitation of enzymes in industrial biotechnology through rational structure-based redesign, and for therapeutic targeting of enzymes by the pharmaceutical industrial. The beneficiaries will initially be academic and industry scientists: enzymologists and biochemistry, spectroscopists, computational chemists and, more generally, those interested in (bio)chemical catalysis.

At the heart of the proposal is the design of experimental and computational data/outputs that can be easily shared with, and benchmarked by, others in the field. The timely sharing of all relevant NMR assignment data, X-ray crystal structures and computational parameters benefits the enzymology, computational chemistry and drug design communities as it facilitates new calculations/simulations and allow rigorous testing and benchmarking of our work.

Exploitation and Wider benefits:
We do not anticipate an immediate commercial impact, given our research is largely focused on fundamental aspects of catalysis by COMT. However, COMT is an established drug target for the treatment of Parkinson's disease, so our work could lead to improvements in drug design. Beneficiaries would initially be Pharma and academics working on drug design. However, if our work leads to improvements in the inhibition (drugging) of COMT, benefits would be felt by the wider community through improved quality of life of e.g. those suffering from Parkinson's disease.

As methyltransferase enzymes have potential uses in biocatalysis as 'green' alkylating catalysts, our work may lead to improvements in the use of enzymes such as COMT in biocatalysis. Beneficiaries would initially be those involved in the design of industrial biocatalysts - academic and industry scientists. However, as industrial biotechnology has the potential to offer complete solutions to major societal challenges such as health, energy supply, food production/security, impact could ultimately be felt by much of society.

As appropriate, we will seek to protect any IP arising from this work at the earliest stage. Our strategy for translating the technology is to establish IP protection through UMIP (Manchester's IP office). We will communicate our progress through networking events with external stakeholders.

Communication and Outreach:
Dissemination of our work to the scientific community will be via timely publication in highly-visible peer-reviewed journals and presentations at leading national and international workshops and conferences.
Due to the prominence of COMT as a major target in the treatment of Parkinson's disease, we expect interest in our work from the wider public. Public engagement activities will initially be embedded into established local (MIB and SYNBIOCHEM) efforts, including annual open days for local A' level students and a number of public engagement events in Greater Manchester including ScienceX and Science Spectacular.
To reach a wider audience in the final year of the project, we will produce of a new 'CAMERA - Chemistry At Manchester Explains Research Advances' video to disseminate key findings from our work to a global online audience.