Exploring the mechanism and scope of the enzymatic formation of five membered ring

Many of todays drugs that we rely on for treatment of cancer, bacterial infection, immune disorders and viral infections are either natural products or are derived from natural products. Natural products remain, even today, a source of drugs and diagnostic molecules. In contrast to man made chemicals natural products are complex in terms of shape and composition. This structural novelty is in part the reason that they work in specific ways, (less side effects). In general, these natural products are made in bacteria rather than in humans or animals. Humans have evolved to ditch much of their complex chemistry. We need vitamins in food, because we cannot make them; rather rely on bacteria or plants to make them and we eat them. Bacteria have an amazing repetoire of complex chemistry that organic chemists can only dream of. In making new man made materials, a key challenge is often to identify plausible new scaffolds or skeletons. Molecules which are easy to draw are hard to make. Yet new scaffolds and motifs (known as chemical diversity) is at the heart of drug discovery. We are going to study the bacterial enzyme that makes heterocyclic amino acids. These five membered rings are a common motif in biologically active compounds but there does not exist any good way of making them in natural products by synthetic chemistry. The use of bacterial enzymes to accomplish chemical tasks is very well known, washing powder being the best known example but they are widespread in the food industry and increasingly the organic chemistry lab. By working out how the enzyme which makes five membered rings works, we will gain control of the enzyme. By doing this we will be able to make novel materials and we believe completely new biologically active compounds. What is more these enzymes work in water at room temperature without producing noxious waste materials.

We propose to investigate the mechanism of the formation of heterocycles by enzyme. These five membered rings are critical components of many important biologically active molecules. We have made significant progress in determining the chemical mechanism, which is the first step to harnessing the enzyme. We intend to pursue the mechanism by novel labeling strategies including stable isotope labeling of substrates. We will also probe the basis of recognition using NMR approaches to identify the crucial residues involved in binding substrate to the enzyme. This is important because in the long term we would wish to replace amino acids by non peptide like groups. We have shown that NMR appears to detect intermediates that form during the reaction. The oxidation state of the rings is important, since even this subtle modification controls activity and stability of the compounds. We have shown that air can be sued to replace in part the enzyme mechanism. We intend to develop further the chemical route but also to study the enzymatic route. There are important and puzzling differences between enzymes that catalyse this reaction. The ability to control the stereocentres of amino acids is also central the activity of these compounds. The mechanism by which residues adjacent to thiazolines are epimerised is unknown. We will determine whether it is spontaneous or enzyme catalysed. We have developed an approach using a combination of peptide synthesis and protein ligation that will allow us incorporate non natural amino acids. This will not only give us exquisite control of the biochemical experiments but lead to more interesting chemical scaffolds in the future.

Academic impact
New tools for scientist interested in natural products. Importantly our work will establish new routes to incorporation of non natural amino acids into biologically active molecules, this will enable scientists in other fields to pursue their use in other challanging problems. The mechanistic and structural insights will transform other scientists ability to manipulate natural product biosynthetic pathways.

Outreach
Naismith is very active in outreach with school children. If funded we will offer summer placements to local children to experience chemical biology. We also intend to create experiments suitable for University undergraduates in the first instance at St Andrews, that could become part of chemistry courses in the UK.

Research and professional skills
The UK has identified synthetic biology and Industrial biotechnology as key deficits in scientists' training for the future workforce. This project is at the cutting edge of such tools and will therefore provide excellent training for Koehnke. Koehnke intends to set up a lab of his own (in Industry or academia) and this project will give him the skills required for this. We will ensure maximum diffusion of these skills by recruting a PhD student to work alongside him but on a defined and distinct project. Researchers will have access to the award winning "Gradskills" courses run by the University of St Andrews, which aim to provide a wide variety of life skills. Naismith runs a summer school for UK and EU graduate students held every two years.

Economic and Societal Impact
The work will generate new biologically active molecules and moreover allow their generation in useful quantities. This means they can be used in drug development programs and in screening cmapaigns. We are setting up collaborations with Industry to maximise their benefit. If funded we can pursue such parnterships. We expect to transfer technology through service agreement (we make compounds) or by technology licence (we have filed a patent). We will consider founding our own spin out company as the project develops.