UC Irvine

Polyketides and fatty acids (FAs) are naturally produced molecules, which are both biosynthesized from acyl-CoA building blocks and have similar enzymatic machinery. However, the structures and functions of polyketides and fatty acids are very different. Polyketides are secondary metabolites that have been used as pharmaceuticals and leads in drug discovery, whereas fatty acids are the components of cell membranes and have roles as signaling molecules. The goal of this dissertation is to gain a better understanding of how the enzymes in type II polyketide synthases (PKSs) and fatty acid synthases (FASs) generate their diverse products.

Type II PKSs generate reactive poly-!-ketone intermediates, which are regiospecifically cyclized and aromatized by aromatase/cyclase (ARO/CYCs) enzymes. In Chapter 2, the structural and functional studies of two functionally divergent di-domain ARO/CYCs are discussed. This work reveals the role of each domain in these di-domain enzymes, and identifies residues in the active site pockets that are important for catalysis.

Type II PKSs typically have an acetyl-starter unit; however, some polyketide natural products exist that contain non-acetate starter units. The incorporation of non-acetate starter unit adds molecular diversity to the poly-!-ketone scaffold. DpsC is the enzyme responsible for the enzymatic incorporation of a propionyl starter unit during daunorubicin biosynthesis. Structural studies of DpsC that reveal insight into its mechanism and substrate specificity are discussed in Chapter 3. AuaEII is the CoA-ligase responsible for the generation of anthranoyl-CoA, which is used as a starter unit during the biosynthesis of a class of hybrid type II polyketides named the aurachins. Chapter 4 discusses the crystal structure of AuaEII and its implications for starter unit specificity of the aurachin biosynthesis pathway. An additional enzymatic strategy to generate structural diversity in type II polyketide natural products is polyketide oxidation. BE-7585A is a type II polyketide natural product that is proposed to catalyze an oxidative rearrangement during biosynthesis. The FAD/NADPH-dependent oxygenase BexE is proposed to catalyze this oxidation. The crystal structure and initial in vitro characterization of BexE are discussed in Chapter 5.

The acyl carrier protein AcpP plays a central role in type II FA biosynthesis in E. coli. AcpP carries the nascent FA chain and delivers intermediates into the active sites of over 12 other enzymes during FA biosynthesis. The protein-protein interactions between AcpP and its target enzymes are transient, and this has hampered efforts to gain information about how AcpP interacts with target enzymes. Chapter 6 describes the application of mechanism-based crosslinkers to generate stable complexes between AcpP and two different partner enzymes: the dehydratase FabA and the ketosynthase FabB. Comparative analysis of the AcpP=FabA and AcpP=FabB crystal structures reveals differential AcpP interactions with partner enzymes during FA biosynthesis. This work serves as a basis for engineering protein-protein interactions to modulate the affinity of AcpP for different partner enzymes and alter fatty acid profiles in vivo for biofuel production.