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The C-glycosyltransferase IroB from Pathogenic Escherichia coli: Identification of Residues Required for Efficient Catalysis

Title:

The C-glycosyltransferase IroB from Pathogenic Escherichia coli: Identification of Residues Required for Efficient Catalysis

Foshag, Daniel, Campbell, Cory and Pawelek, Peter D. (2014) The C-glycosyltransferase IroB from Pathogenic Escherichia coli: Identification of Residues Required for Efficient Catalysis. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1844 (9). pp. 1619-1630. ISSN 1570-9639

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Official URL: http://www.sciencedirect.com/science/article/pii/S...

Abstract

The E. coli C-glycosyltransferase IroB catalyzes formation of a C-C bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65-67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Foshag, Daniel and Campbell, Cory and Pawelek, Peter D.
Journal or Publication:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Date:September 2014
Funders:
  • NSERC
ID Code:981280
Deposited By: PETER PAWELEK
Deposited On:19 May 2016 17:56
Last Modified:18 Jan 2018 17:52
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