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

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.