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Identification of a Surface Glutamine Residue (Q64) of Escherichia coli EntA Required for Interaction with EntE

Title:

Identification of a Surface Glutamine Residue (Q64) of Escherichia coli EntA Required for Interaction with EntE

Khalil, Sofia, Jaworski, Ian and Pawelek, Peter D. (2014) Identification of a Surface Glutamine Residue (Q64) of Escherichia coli EntA Required for Interaction with EntE. Biochemical and Biophysical Research Communications, 453 (3). pp. 625-630.

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Abstract

The enterobactin biosynthetic enzyme EntA forms a complex with EntE, the next enzyme in the pathway, to enhance activation of the enterobactin precursor 2,3-dihydroxybenzoate. Here we used phage display to identify an EntE-interacting region on the surface of EntA. Upon panning immobilized EntE with a random peptide phage library, we recovered 47 unique EntE-binding dodecamer peptide sequences that aligned to a region of the EntA primary sequence corresponding to helix α4. In order to further investigate this region, we mutagenized EntA Q64, a hydrogen-bonding residue found on the surface-exposed face α4. Far-UV circular dichroism, thermal denaturation experiments, and enzymatic assays showed that mutation of EntA residue Gln 64 to alanine (Q64A) had no deleterious effect on EntA structure or function. By following near-UV CD spectral changes, we found that the spectrum of wild-type EntA was altered in the presence of EntE, indicative of conformational changes in EntA aromatic chromophores upon formation of the EntA-EntE complex. However, EntE did not affect the CD spectrum of EntA variant Q64A, demonstrating that this variant did not interact with EntE in a manner similar to wild-type EntA. Analytical ultracentrifugation of wild-type and variant EntA proteins showed that EntA Q64A was predominantly dimeric at 20 μM, unlike wild-type EntA which was predominantly tetrameric. Taken together, our findings establish that EntA α4 is required for efficient formation of the EntA-EntE as well as for EntA oligomerization.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Khalil, Sofia and Jaworski, Ian and Pawelek, Peter D.
Journal or Publication:Biochemical and Biophysical Research Communications
Date:24 October 2014
Funders:
  • NSERC
Digital Object Identifier (DOI):10.1016/j.bbrc.2014.09.131
ID Code:981277
Deposited By: PETER PAWELEK
Deposited On:19 May 2016 17:55
Last Modified:18 Jan 2018 17:52
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