Abstract

To obtain scarce iron from their environment, bacteria synthesize and secrete high affinity siderophores that chelate ferric ions. Enterobactin is a catecholate siderophore produced by Escherichia coli. Synthesized in the cytoplasm by the concerted action of seven enzymes, EntCBDAEF and EntH, enterobactin is then transported to the periplasm by the inner-membrane protein EntS. We hypothesize that the Ent proteins assemble in a large multiprotein complex to enhance the metabolic flux of the intermediates and prevent their diffusion, and that this assembly localizes at the inner membrane where it interacts with EntS for secretion. The overarching goal of this thesis is to gather further structural evidence of interactions among the biosynthetic enzymes, to determine the manner in which the proposed assembly interacts with the transporter channel, and to initiate characterization of EntS. Herein we demonstrate an interaction between the first two enzymes of the pathway, EntC and EntB. Through in vivo crosslinking with formaldehyde, we captured intracellular complexes comprising both proteins. These results were supported by two-hybrid assays, whereas automated docking of the crystal structures of EntC and EntB resulted in a model where the active sites of EntC and EntB are oriented in apposition and connected by an electropositive surface potentially capable of channeling the negatively charged precursor of enterobactin, isochorismate. We then built on these research outcomes, which suggested that other Ent proteins could interact simultaneously and form complexes in which both EntC and EntB were detected. Using in vivo crosslinking with formaldehyde, we confirmed the presence of proteins EntA and EntE in these higher-order complexes. We also initiated work towards identification of EntS cytosolic interactors, where using an engineered ascorbate peroxidase enzyme fused in frame with EntS as the bait, proximity labeling assays revealed prey proteins localizing within 10 nm of EntS. Finally, we undertook extraction and isolation of EntS in a detergent micelle and in synthetic lipid nanodiscs. We identified two detergents and two copolymers that were efficient in extracting and solubilizing EntS, as well as the optimized conditions for successful extraction. The results obtained also suggest that EntS might assemble as a dimer.