Studies of the Protein Interaction Network Required for Enterobactin Biosynthesis in Escherichia coli

• Main Author: Khalil, Sofia
• Format: Others
• Published: 2011
• Online Access: http://spectrum.library.concordia.ca/7531/1/Khalil_Phd_S2011.pdf; Khalil, Sofia <http://spectrum.library.concordia.ca/view/creators/Khalil=3ASofia=3A=3A.html> (2011) Studies of the Protein Interaction Network Required for Enterobactin Biosynthesis in Escherichia coli. PhD thesis, Concordia University.

Abstract Studies of the Protein Interaction Network Required for Enterobactin Biosynthesis in Escherichia coli Sofia Khalil, Ph.D. Concordia University, 2010 Siderophores are small-molecule iron chelators that many bacteria synthesize and secrete in order to survive in iron-depleted environments. In Escherichia coli, biosynthesis of the siderophore molecule enterobactin requires the activities of six enzymes, EntA-EntF. These enzymes function sequentially to produce enterobactin molecule in the cytoplasm. The enterobactin biosynthesis pathway is divided into two modules. The first module involves the conversion of chorismate to 2,3-dihydroxybenzoic acid (2,3-DHB), and requires the activities of EntC, EntB (N-terminal domain) and EntA. The second module involves non-ribosomal peptide synthesis (NRPS) such that three molecules of 2,3-DHB are condensed with three molecules of L-serine to form the siderophore. The NRPS module requires the activities of EntE, EntB (C-terminal domain), EntD and EntF. The overall goal of my research project is characterization of the enterobactin biosynthetic enzyme EntE. EntE catalyzes the activation of 2,3-DHB via adenylation producing DHB-AMP. I am interested in addressing the following questions: (i) How does EntE bind its 2,3-DHB substrate? (ii) How does it interact with its upstream and downstream partner proteins: EntA, which produces 2,3-DHB, and EntB, which uses the EntE product (DHB-AMP) as a substrate, respectively? My thesis is divided into three research-related chapters: The first research chapter focused on the interaction of EntE with its substrate, 2,3-DHB, as well as the characterization of EntE-EntB interaction in the presence and absence of 2,3-DHB. A significant change in EntB conformation was observed upon the interaction with EntE when in the presence of 2,3-DHB. In the pull-down assay, EntB as bait protein pulled down more EntE in the presence of exogenous 2,3-DHB. We conclude from this chapter that the ligand-loaded state of the protein was necessary for efficient protein-protein interaction. The second research chapter involves the characterization of a novel interaction between EntE and its upstream partner protein EntA. A significant increase in EntE activity was observed upon adding EntA. Furthermore, EntA reduces the FRET signal of EntE-bound 2,3-DHB in a saturable manner with increasing EntA concentrations. Using this fluorescence binding assay at 20 °C revealed a positive cooperativity in EntA-EntE interaction with Hill coefficient greater than one. The AUC experiments showed that EntA conformation is highly dependent on its concentration. In conclusion, the results of this chapter suggest that EntA-EntE interaction likely induces remodeling of EntE active site, resulting in the observed increase in EntE catalysis. In the third research chapter, the EntA-EntE interaction interface was characterized using phage display. Based on the interaction interface predicted by phage display data, EntA variants containing single-site and double-site mutations were created (Q64A, A68Q, and Q64A/A68Q). Our in vitro biophysical techniques and growth phenotype experiments revealed that EntA (Q64A) and EntA (Q64A/A68Q) mutations have a more pronounced effect than EntA (A68Q) mutation on disruption of the EntA-EntE interaction interface.