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PMID: 15598734 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

In vitro characterization of IroB, a pathogen-associated C-glycosyltransferase.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 102 ·No. 3 ·2005-01-18 ·Pages 571-6

Fischbach MA, Lin H, Liu DR, Walsh CT

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.

MeSH Terms
Cloning, Molecular/methods Enterobactin/metabolism Escherichia coli Proteins/chemistry,genetics,metabolism Glucosyltransferases/chemistry,genetics,metabolism Glycosylation Glycosyltransferases/chemistry,genetics,metabolism Kinetics Uridine Diphosphate Glucose/metabolism
Chemicals
Escherichia coli Proteins Enterobactin Glycosyltransferases Glucosyltransferases IroB protein, E coli Uridine Diphosphate Glucose
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Fischbach Michael A
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Lin Hening
Liu David R
Walsh Christopher T
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0408463102
Published
2005-01-18
Epub
2004-00-14
Pages
571-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC545562
Subset
IM
Grants
NIGMS NIH HHS · F32 GM020011 · United States
NIGMS NIH HHS · GM065400 · United States
NIGMS NIH HHS · GM20011 · United States
NIGMS NIH HHS · R01 GM065400 · United States
NIGMS NIH HHS · R01 GM020011 · United States
Corrections
CommentIn
PMID: 15647344
External Links
PubMed source DOI: 10.1073/pnas.0408463102 PMC Full Text View journal details
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