| Popis: |
Nonribosomal peptide synthetases (NRPSs) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. Each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. Central to each module is the carrier protein (CP), which is first primed via attachment of a 4′-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. The ppant arm then covalently harbors activated monomers and growing peptides and shuttles them between the active sites of catalytic domains in both the same and adjacent modules. During CP priming and peptide elongation, a CP thus exists in multiple different post-translational states and must interact with multiple catalytic domains. Understanding how NRPSs are able to efficiently orchestrate this series of sequential protein-protein interactions between a CP and its partner catalytic domains is key to understanding the molecular mechanism of NRP synthesis. In functionally analogous fatty acid synthases (FAS) and polyketide synthases (PKS), the post-translational state of a CP (holo vs. substrate loaded) has been implicated in directing the sequence of interactions in these systems. However, the role these modifications play in modulating protein-protein interactions in a NRPS has not previously been explored. Here, we provide evidence that the biochemical state of a CP (apo vs. holo vs. monomer loaded) alters the affinity of the CP for its partner catalytic domains in a NRPS system. Our results demonstrate that each partner catalytic domain preferentially binds to a different biochemical state of the CP and suggests a means by which directionality in protein-protein interactions is achieved in NRPSs. |
