Biosynthesis of five-membered heterocyclic rings

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.