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Developing Chemical Biology Tools to Study and Engineer Fatty Acid Synthases


Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. The interactions between ACP and catalytic partner enzymes are transient in nature and extremely difficult to visualize. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. The toolbox available to modify ACP and study the intricacies of the FAS has grown in the past decade, speicifcally with AcpH technology to produce large quantities of apo-ACP and “one-pot” reaction protocols to modify apo-ACP with almost any substrate including fluorescent moieties, substrate mimics or inhibitors.

This dissertation focuses on the further expansion of the ACP modifying toolkit and applications of this toolkit. A mechanism-based crosslinking probe with a 3-alkynyl-sulfone pantetheine scaffold was designed and synthesized to covalently tether the active site of dehydratase (DH) domains and ACP. Two different versions of this probe were applied to E. coli proteins to tether AcpP with both DHs, FabA and FabZ. The AcpP=FabA and AcpP=FabZ complex crystal structures were solved to 1.9Å and 3.4Å respectively, and were the first two crystal structures of a native FAS system where the ACP was caught interacting with a partner protein. By analysis of the crystal structure, NMR studies, and mutagenesis studies, the mechanism of substrate translocation from the AcpP hydrophobic pocket to the FabA substrate pocket was elucidated. By applying varied chain length mechanism-based probes and a newly designed crosslinking assay, “positive-patch” residues on FabA and FabZ and “gate-keeping” residues at the front and back of the substrate binding tunnel were shown to be important for both protein-protein interactions and substrate chain length preference.

The enoyl reductase (ER) domain is the rate-limiting step in FAS and has long been of interest as an antibiotic target, but lacks detailed structural information. We have developed a non-covalent crosslinking probe for the first time based on a prototypical tight-binding inhibitor of ER (FabI in E. coli), triclosan. When appended to AcpP, the triclosan probe was shown to inhibit FabI in the μM range, pull FabI exlusively out of cell lysate, and have nm binding affinity, stabalizing the complex for future crystallography studies. Finally, we utilized the acyl-acyl carrier protein synthetase (AasS) from V. harveyi to modify AcpP both in vitro and in vivo with unnatural cargo including azide, alkyne and phenyl moieties. This technology allows for facile access to unique ACPs with a natural thioester linkage to study ACP activity in the FAS. Applications also extend in vivo for chemical production or direct fluorescent imaging of FAS products in the cell.

These new ACP modifying tools allow for the visualization of ACP and partner protein interactions that lend insight into how this elaborate assembly line functions. Additionally, these tools have broad applications in bioorthogonal labeling for imaging and synthetic biology applications. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory.

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