UC San Diego

Fatty acid synthases (FASs) iteratively condense and reduce two carbon ketide units to produce fatty acids, which serve as precursors utilized in membrane development and homeostasis, energy storage, cofactor biosynthesis, and signaling. The core enzymatic activities of FASs are conserved, but they can exist as either large multidomain megasynthases (type I) or as diffuse monofunctional proteins (type II). Central to FAS is the small, 8kD acyl carrier protein (ACP), which carries thioester-linked pathway intermediates

to each respective enzyme active site. Each FAS enzyme, or domain, must form productive protein-protein interactions (PPIs) with ACP in addition to recognizing the ACP-tethered cargo. The robust and modular biosynthetic logic of FASs, and the evolutionarily related polyketide synthases (PKSs), make these synthases attractive metabolic engineering platforms to produce either industrially relevant feed stocks or novel bioactive compounds. Unfortunately, a fundamental lack of knowledge regarding ACP-mediated PPIs has led to limited successes in combinatorial engineering efforts.

Here we use the Escherichia coli type II FAS as a model system to investigate ACP-mediated PPIs and substrate processing by two important FAS enzymes, ketosynthases (KSs) and malonyl-CoA ACP transacylases (MATs). The first chapter provides an up-to-date review of ACP-mediated PPIs in E. coli type II FAS. In the second and third chapter we use this well-characterized system to better understand ACP-KS PPIs and KS substrate discrimination by structurally characterizing the E. coli elongating KSs, FabB and FabF, as substrate/intermediate complexes with ACP that approximate states formed during catalysis. In conjunction with molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational changes mediated by two active site loops that regulate substrate processing. Additionally, we developed three assays, one in vitro and two in vivo, to systematically evaluate and characterize the ACP-KS interface.

In the fourth chapter, we investigate PPIs between E. coli AcpP and its cognate MAT, FabD. We solved a high-resolution crystal structure of AcpP in complex with FabD and thoroughly interrogate this system using enzyme assays and molecular dynamics simulations. Results from these studies demonstrate that interfacial plasticity at the AcpP-FabD interface serves to facilitate rapid FabD transacylation rates.