Structural and functional characterization of enzymes promising biocatalytic prowess

Abstract

Natural products are a diverse class of molecules possessing various different properties — including medicinally relevant bioactivities. In hope to find and develop new drugs, researchers and health care providers are turning more frequently to molecules not obeying to Lipinski’s infamous rule of five. Such molecules, usually significantly larger than canonical small molecule drugs are able to manipulate different targets compared to traditional small molecule drugs. A large class of these formerly ‘undrugable’ targets are protein-protein interactions (PPIs). An orchestra of molecules affecting these PPIs could be used to tune cellular and tissue biochemistry aiding in disease treatment or symptom management. The synthesis of these molecules can be challenging, regardless of approach, due to their size and wealth of different chemical functionalities. For reasons mentioned before, cyclic peptide natural products have been shown to posses a wide range of medically relevant bioactivities. One example is the FDA approved immunosuppressant cyclosporine A. Cyclic peptides are particularly valuable for drug development, due to their conformationally restrained structure, resistance to proteases and oral bioavailability (the preferred route of drug administration). Fighting entropy, this cyclisation reaction can be difficult to achieve depending upon the peptide backbone. However, Nature has found means for macrocyclisation, which can be employed in vitro accessing cyclic peptides comprised of canonical and non-canonical amino acids, allowing access to a wide range of novel man-made cyclic peptides with a range of putative novel bioactivities. On the down side, these biocatalysts/enzymes have either been too slow to produce large cyclic peptide quantities, or been displaying a substrate scope too narrow. In this thesis the novel macrocyclase PCY1, which is involved in the biosynthesis if so called segetalins, is functionally and structurally characterised, allowed us to assess its potential for biocatalytic applications. Based upon these fundamental enquiries, a system is devised allowing access to a wider range of cyclic peptides, whilst compensating for atom-economic disadvantages. Furthermore the structural investigation of a new halogenase with a natural preference towards iodine is presented as well. Biocatalytic and thus greener access to CH activation reactions is crucial for pivotal cross-coupling reactions, now widely applied in organic synthesis, accessing a vast complexity of known and novel chemical structures.