Synthetic Substrate Analogues as Tools for Structural Studies of Protein-Protein and Protein-Substrate Interactions in Microbial Biosynthesis

Fatty acid synthases (FAS) and polyketide synthases (PKS) are closely related biosynthetic enzymes that form large, multi-domain enzyme complexes. FASs are a pillar of primary metabolism across all domains of life. These enzymes are responsible for the biosynthesis of the various fatty acid chains that comprise cellular membranes. PKSs are an important component in the secondary metabolism of many organisms. These enzymes biosynthesize a wide variety of bioactive natural products with many pharmaceutical and industrial applications. Both FASs and PKSs are widely studied as platforms for bioengineering, biofuel production, and biocatalysis.;One of the primary issues in the field is the difficulty of studying the protein-protein interactions in these pathways due to the transient nature of the interactions. This knowledge gap has greatly limited the effectiveness of combinatorial biosynthesis. Another primary issue in the field is the extreme reactivity of the enzymatic substrates and biosynthetic intermediates, which makes studying the protein-substrate interactions in these pathways nearly impossible. The lack of this information has hampered rational engineering of enzymatic active sites. The goal of this work is to overcome these obstacles through the utilization of synthetic substrate analogues. These analogues were designed to trap otherwise transient protein-protein and protein-substrate complexes for X-ray crystallographic study.;In this dissertation, the application of a mechanism-based crosslinker is described which enabled the first structure determination of an acyl carrier protein (ACP) - ketosynthase (KS) complex from E. coli. This crystal structure, which is the only available structural data on the ACP- KS interface, was then used to design surface mutants of the KS that altered the fatty acid profile of E. coli cultures. This is the first reported case of successfully leveraging protein-protein interactions to modulate the product profile of a metabolic pathway. Also in this dissertation, the use of a polyketide substrate mimic is presented which allowed for the first structure determination of an acyl-KS intermediate in complex with an extender unit analogue. This crystal structure provided the only snapshot of a KS "in action" with all substrates positioned for catalysis and enabled a mechanism for the enzyme's unique activity to be proposed.