The biosynthetic logic of polyketides synthases (PKS) and fatty acid synthases (FAS) are similar in utilization of acyl-CoA building blocks, biosynthetic enzymes and chemical reactions yet they yield vastly different products. While polyketide is a group of secondary metabolites with diverse and complex chemical structures and biological activities, fatty acid structures are simple and comprised of fully reduced aliphatic chains. A central question underlying PKS and FAS research is how these parallel systems lead to the production of these two structurally and functionally divergent types of metabolites.

The focus of this dissertation is the dissection of PKS and FAS structures and functions to gain insights into sequence, structure and function relationships of PKS and FAS enzymatic domains. Particularly, this work is focused on 1) the dissection of the trans-acting enoylreductase, LovC, its catalytic mechanism and how its substrate and cofactors specificities are promoted, 2) the understanding of the protein-protein and protein-substrate interactions between the β-hydroxy-acyl carrier protein (ACP) dehydratase, FabA, and its native ACP, AcpP, and 3) the engineering of LovC and FabA altered substrate specificities.

Structural analysis of LovC coupled with docking studies, site-directed mutagenesis and in vitro activity assays are reported. Our results illustrate a decrease in activity with the K54A mutant allowing for proposed role of the active site residues and elucidation of the catalytic mechanism. We also identified residues important for substrate and cofactor specificities with mutations that preclude the enzyme's ability to competently bind substrate analogs and NADPH. Importantly, we demonstrate that mutation of N263 allowed for alteration of substrate specificities.

The first crystal structure of a di-domain fatty acid synthase enzyme with ACP is reported, the crosslinked crystal structure of AcpP=FabA. The ternary complex displays 2AcpP:2FabA:2 crosslinker probe stoichiometry with a dimer interface along the two β7 of the two FabA. Importantly, the structure illustrates significant structural and dynamic asymmetry between the two AcpP-FabA interfaces. This asymmetry combined with NMR and accelerated molecular dynamics (AMD) provided an unprecedented animated view of the interactions between AcpP and FabA. This work also demonstrates that crosslinking allowed for capturing of ACP in functional association with catalytic domains. As such, we developed an in vitro crosslinking activity assay to test the function of FabA mutants. Through single point mutations, we engineered FabA to alter its substrate specificities towards short carbon chain lengths, mutants with promiscuous activities towards both short and long carbon chain lengths and a mutant with an extended substrate tunnel with the capacity to bind extended fatty acid chain lengths.