UC San Diego

Carrier protein (CP) dependent biosynthetic pathways are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their natural products. These pathways, which include the fatty acid synthase (FAS), polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS), have been targeted for engineering through substitution of modules, domains and subdomains. This method, termed combinatorial biosynthesis, has been met with limited success due to the lack of proper protein-protein interactions between noncognate proteins. With catalysis mediated by specific protein-protein interactions between the carrier protein and its partner enzymes, enzymology and control remain fertile ground for discovery. Here, I investigate the biomolecular recognition between the peptidyl carrier protein (PCP) and adenylation (A) domains of type II NRPS systems as my first step in engineering these synthases. The first chapter provides a recent review of the structural biology of transient NRPS PCP and partner protein complexes to identify the specific modes of PCP recognition in the type I and type II NRPS. The second and third chapter presents a thorough structural analysis of the PCP-A domain protein-protein interface from prodigiosin and pyoluteorin biosynthesis. The PCP-A domain complexes were stabilized using a mechanism-based inhibitor, which afforded crystallization and successful structure determination of two cognate and one noncognate PCP-A domain complexes. This high-resolution information was integrated with previous NMR titration data, MD simulations, and mutagenesis studies to reveal PCP dynamics and specific protein-protein interactions that govern PCP-A domain complex formation. The fourth chapter involves application of the previously solved PCP-A domain structures towards a development of a computational protein-protein interface design protocol to create a hybrid natural product pathway. The first PCP-A domain structure solved in this work was used towards computational design of a new interface between the acyl-carrier protein (ACP) from Escherichia coli fatty acid biosynthesis. The optimized computational design protocol was able to improve noncognate A domain activity by ~1600 fold through the manipulation of electrostatic interactions to create a new protein-protein interface. Through these chapters, I have proven that the coupling of biophysical data to computational methodologies can be the next platform towards re-engineering of carrier-protein dependent pathways to create novel natural products.