Chemoenzymatic investigations of protein-protein interactions in carrier protein-mediated biosynthesis - eScholarship

The carrier protein is an essential component in a variety of evolutionarily distinct biosynthetic pathways. In these systems, the carrier protein is responsible for interacting with enzyme activities contained in multi- enzyme complexes of varying architecture. Deciphering the mechanisms of these interactions is important for our complete understanding of these highly specialized systems. New methods for determining functional interactions between enzymes involved in carrier protein- dependent biosynthetic pathways are necessary to achieve this goal. We have developed techniques to assess the interactions between carrier protein and ketosynthase (KS) enzymes from fatty acid and polyketide synthases, and, more recently, between the carrier protein and the adenylation domain from nonribosomal peptide synthetase. Both methods are derived from the known promiscuity of the CoA biosynthetic pathway and the phosphopantetheinyl- transferase (PPTase) Sfp, both of which are involved in the post-translational modification of the carrier protein. Herein, we describe a mechanism-based crosslinking method to covalently tether an ACP to its cognate ketosynthase enzyme, in a manner that functionally mimics the normal transfer reaction between substrate- bound ACP and the active site of the KS. Using this method, we have shown it to be amenable to the four classes of biosynthetic architecture that include these activities - both type I and type II fatty acid and polyketide synthases. In addition, modifications of this approach using a panel of reagents of varying sizes and functionality have afforded us a method to assess the substrate specificity of these KS enzymes. Finally, we have used this method and a recently developed adenylation assay to determine the contribution to functional interactions with partner enzymes of specific regions of the carrier protein. The mechanism-based crosslinking method is useful for its potential to yield natural CP-KS co-crystal structures for all four fatty acid and polyketide synthase architectures. Both this method and the adenylation assay enable us to determine the compatibility of carrier proteins in unnatural systems. This research will be important for the development of combinatorial biosynthetic systems and the future synthesis of novel natural products for pharmaceutical applications