Crosslinking studies in natural products biosynthesis

Many pharmaceuticals on the market today belong to a large class of natural products called nonribosomal peptides (NRPs). Responsible for their biosynthesis are multimodular enzymes, called nonrbisomal peptide synthetases (NRPSs). They employ the carrier protein to mediate proper processing of biosynthetic intermediates by tethering monomeric units as they are condensed and modified by other catalytic enzymes in the synthetase. Biochemical and genetic investigations of these enzymes have uncovered the key principles of NRP synthesis; however, little progress has been made in the manipulation of this biosynthetic machinery for the production of novel therapeutic agents due to what little is known about the protein interactions governing NRP biosynthesis. As a result, new approaches are needed to uncover the structural and mechanistic features involved in the selective communication between these enzymes.;We have developed novel chemical probes capable of facilitating structural and mechanistic studies of the dynamic intra- and intermodular interactions within NRPSs. Employing bioorthogonal probes compatible with carrier protein modification, we designed a crosslinking assay for examining protein interactions between cognate partner NRPSs. In this study, we demonstrated that the probes capable of crosslinking NRPS modules with compatible communication (COM) mediating domains under copper free conditions serve as unique diagnostic tools for exploring protein interactions in NRPS systems. We also synthesized a mechanism-based probe of the epimerase (E) domain, adding another chemical tool to our arsenal of crosslinking probes. This study showed that chlorovinylglycine acts as an irreversible inactivator of the E domain utilizing a condensation assay dependent on E domain activity. Given that the bioorthogonal crosslinking probes as well as the mechanism-based probe of the E domain rely on the carrier protein for functional activity, these probes serve as ideal chemical tools for engaging in structural studies to decipher the molecular mechanism and chemical biology underlying the discrete domains within not only NRPS systems but also other carrier protein-mediated biosynthetic assembly lines.