Many medically relevant peptides are naturally produced by non-ribosomal peptides synthases (NRPSs); however, there are challenges to understand their production due to the covalently attachment of the biosynthetic intermediates to the NRPS enzyme. Hence, it is crucial to investigate this process to further redesign the biosynthetic pathways and consequently, produce new potent compounds. A common feature in any NRPS biosynthesis is the pantetheine linker between the NRPS machinery and the substrate. Here, we aim to develop and utilise novel chemical biology tools around this linker to gain a better understanding of the biosynthesis of NRPS-produced antibiotics.
A series of modified peptidyl-coenzyme A probes (coenzyme A (CoA) being the precursor loaded onto the carrier proteins that affords the pantetheine linker), have been chemically synthesised using solid phase peptide synthesis (SPPS) and loaded on to the PCP-X di-domain from the NRPS machinery.1 Subsequently, monooxygenase enzyme activity has been tested in vitro using these probes as potential substrates. The probes are designed based on the linear peptide structure of vancomycin with the modification either at the C-terminus or the N-terminus, and varying peptide length. The results of these experiments reveal the tolerance of the monooxygenase enzymes of the vancomycin machinery to accept these modified peptidyl-CoA probes, which is important for the future generation of modified antibiotics. Furthermore, we will further develop these probes by modifying the part of the pantetheine linker to gain structural insights into this complex NRPS system.