Abstract

Iron is an indispensable micronutrient for most microorganisms. A high-affinity iron chelator called enterobactin is synthesized by Escherichia coli to scavenge scarce ferric iron from its extracellular environment. The biosynthesis of enterobactin is carried out by sequential orchestration of six enzymes, EntC, EntB, EntA, EntE, EntF, and EntD. Previous studies on enterobactin biosynthesis and other siderophores, like pyoverdine and bacillibactin, indicate protein-protein interactions (PPIs) are extensively engaged in these processes, leading to the thesis research goals of ongoing mapping and functional characterization of this PPI. We hypothesize these interactions, albeit weak, may occur among EntC, EntB, and EntA which are responsible for synthesizing 2,3-dihydroxybenzoate (2,3-DHB), a precursor for enterobactin. Furthermore, we hypothesize that a higher-order enzymatic assembly (i.e., a metabolon) forms in the E. coli cytoplasm to facilitate efficient catalysis and regulation of iron homeostasis at the protein level. Here the first evidence of an interaction between EntC and EntB both in vivo and in vitro is reported, thereby completing the map of pairwise PPIs among all the enterobactin biosynthetic enzymes. Further characterization reveals an electropositive channeling surface between the EntC active site and EntB isochorismate active site, suggesting an electrostatic channeling mechanism of enhancing metabolic flux by preventing labile intermediates from diffusing into the bulk solution. Given EntB’s central role in enterobactin biosynthesis, this enzyme is likely a target for regulation. A novel role for 2,3-DHB as a feedback inhibitor of EntB isochorismatase activity was identified, as well as its effect on the down-regulation to the EntCB isochorismate channeling. Finally, a chimeric protein was constructed, composed of dimeric and enzymatically active EntB and EntA fused components, paving the way to investigate the potential existence of higher-order Ent complexes and their recognition of cognate partners.