In Vitro Characterization of IroB, a Pathogen-Associated C-glycosyltransferase

Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):571-6. doi: 10.1073/pnas.0408463102. Epub 2004 Dec 14.

Abstract

Pathogenic strains of Escherichia coli and Salmonella enterica modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the iroA locus [Hantke, K., Nicholson, G., Rabsch, W. & Winkelmann, G. (2003) Proc. Natl. Acad. Sci. USA 100, 3677-3682]. Here, we report the purification of the IroB protein and its characterization as the Ent C-glucosyltransferase. IroB transfers glucosyl groups from uridine-5'-diphosphoglucose to C5 of one, two, or three of the 2,3-dihydroxybenzoyl units of Ent to yield monoglucosyl-C-Ent (MGE), diglucosyl-C-Ent (DGE), and triglucosyl-C-Ent (TGE). DGE, also known as salmochelin S4, and macrolactone-opened derivatives have been isolated from the culture broths of S. enterica and uropathogenic E. coli [Bister, B., Bischoff, D., Nicholson, G. J., Valdebenito, M., Schneider, K., Winkelmann, G., Hantke, K. & Sussmuth, R. D. (2004) Biometals 17, 471-481], but MGE and TGE have not been reported previously. IroB has a k(cat) of approximately 10 min(-1) for the first C-glucosylation and is distributive, with sequential conversion and buildup of MGE and then DGE. The C5 to C1' regio-selectivity of the 2,3-dihydroxybenzoyl-glucose linkage at all three rings of TGE suggests a C5 carbanion, para to the C2 phenolate oxygen, as the carbon nucleophile in this novel enzymatic C-glucosylation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cloning, Molecular / methods
  • Enterobactin / metabolism
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Glucosyltransferases / chemistry*
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism
  • Glycosylation
  • Glycosyltransferases / chemistry*
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism
  • Kinetics
  • Uridine Diphosphate Glucose / metabolism

Substances

  • Escherichia coli Proteins
  • Enterobactin
  • Glycosyltransferases
  • Glucosyltransferases
  • IroB protein, E coli
  • Uridine Diphosphate Glucose