The enzymatic methylation of the peptide bond

Amino acids are nature's open source technology which can be linked together to build machines and it is increasingly realised, potential drug molecules. There are twenty natural amino acids and these have very different chemical properties, shapes and sizes. Yet they connect together using the common peptide or amide bond. Peptide molecules, in theory, could make very good drugs because one can vary the amino acid very easily to tune it for the particular need yet linear strings of amino acids are not good drugs. They are too easily degraded, they do not go into cells and they are too flexible to bind strongly. Nature has evolved a solution to all these problems. To make the macrocycle go into cells it modifies the amino acids within by adding a methyl group onto the amide. This modification would be enormously useful if we could harness it and use in the lab. However, the modification is very difficult to accomplish chemically. This is because the amide which links together amino acids is very stable and unreactive; it is precisely because it is so unreactive that proteins are natures machines. This is yet another example of nature finding an elegant solution to difficult problem. sWe have a strong track record in engineering enzymes to do difficult chemistry, but key to this is understanding the enzymes. In this grant, we seek to understand a 'methylator'. With this information in hand, we will engineer an enzyme to be used both in the lab and inside cells to make starting points for new medicines

Linear peptides do not make useful drugs because they are unstable, conformationally flexible and do not penetrate cells. Peptides, however, make excellent in vitro probes because they are so easily diversified around a common scaffold; the amide bond. Highly modified cyclic peptides are a common motif in potent biologically active natural products. There are examples of such products active in almost all major therapeutic areas. The biosynthetic pathways are attractive as catalysis and recognition are often spatially separate allowing their use in biotechnology. Here we propose to study the addition of a methyl group from S-adenosyl methionine, a modification of peptide macrocycles that is essentially completely uncharacterised. The enzyme that methylates the amide nitrogen in a peptide was only discovered this year; this is an unprecedented chemical modification at room temperature. Methylation of the amide nitrogen is known to greatly increase the cell permeability of macrocycles and this understanding has significant biotechnological potential.

The project will have impact in academia, biotechnology, public understanding of science and in training of scientists for the UK economy.

Academic impact
The project will change how chemists think about activating amides within proteins, this is a reaction of the widest interest. We expect the project will lead to highly cited original research and are committed to ensuring the maximum impact, therefore will use open access publishing and deposit data in public databases. The PI is invited to between five and ten national and international meetings to speak each year, these provide a venue to ensure the work is high profile. In addition to publication, we will deposit the genes we create in ADDGENE to ensure the maximum impact in the community.

Biotechnology
The UK has Europe's strongest biotechnology sector. The synthetic biology in this grant has applications in both cell factory type approaches as well as in vitro chemo enzymatic processes. We have a clear plan for the PI and the co-workers to engage with commercial and academic biotechnological networks. This engagement at such meetings will start at once and is different from the normal academic conference or publication.

Outreach
We will ensure that the general public, particularly children, will benefit from outreach activities that will be undertaken. The PI has a good track record in public engagement and gives public lectures, visit schools and engages with science festivals. In this grant, we have planned and costed the production of hands on exhibits that can be taken into schools. The science in the grant can be made at different levels. For the youngest children, they can build simple models that show the rigidity that comes from making macrocycles and showing the uses of them in medicine. For older children with more chemical knowledge, the 'hard to do' chemistry that enzymes can accomplish at room temperature in water leads to a discussion of green technologies. Since one of our goals is the biotechnological exploitation, there is an opportunity to show how basic science can be translated to economic benefit.

Research and professional skills
The project involves a mixture of techniques; organic chemistry, structural biology, protein engineering, analytical biochemistry, biotechnology, and biophysics. This a very desirable grounding for younger scientists; no job in the UK requires a single skill. Sixteen post-doctoral co-workers have gone onto independent research careers and all co-workers remain in science. If the grant is funded, the PI will recruit at least one PhD to work in a related project alongside the post-doctoral researchers. The PI has mentored twenty-two PhD students who have gone on to graduate and this project has many opportunities for such training.

Economic and Societal Impact
We are committed to ensuring there is an economic impact of this research. We have experience in setting up a spin-out company. We are working with the Oxford University technology transfer office to reach out to potential funders or commercial partners.