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Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE

Title:

Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE

Pakarian, Paknoosh and Pawelek, Peter D. (2016) Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE. Biochemical and Biophysical Research Communications, 478 (1). pp. 25-32. ISSN 0006291X

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Official URL: http://dx.doi.org/10.1016/j.bbrc.2016.07.105

Abstract

Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB- phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Pakarian, Paknoosh and Pawelek, Peter D.
Journal or Publication:Biochemical and Biophysical Research Communications
Date:15 July 2016
Funders:
  • Natural Sciences and Engineering Research Council of Canada (NSERC)
  • Le Fonds de recherche du Québec – Nature et technologies (FRQNT) PBEEE/MELS Scholarship
Digital Object Identifier (DOI):10.1016/j.bbrc.2016.07.105
ID Code:985056
Deposited By: PETER PAWELEK
Deposited On:08 Apr 2019 15:24
Last Modified:08 Apr 2019 15:25
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