Re-evaluation\nof the <i>C</i>‑Glucosyltransferase\nIroB Illuminates Its Ability to <i>C</i>‑Glucosylate\nNon-native Triscatecholate Enterobactin Mimics

The pathogen-associated <i>C</i>-glucosyltransferase\nIroB is involved in the biosynthesis of salmochelins, <i>C</i>-glucosylated derivatives of enterobactin (Ent), which is a triscatecholate\nsiderophore of enteric bacteria including Salmonella\nenterica and Escherichia coli. Here, we reassess the ability of IroB to <i>C</i>-glucosylate\nnon-native triscatecholate mimics of Ent, which may have utility in\nthe design and development of siderophore-based therapeutics and diagnostics.\nWe establish TRENCAM (TC) and MECAM (MC), synthetic Ent analogs with\ntris(2-aminoethyl)amine- or mesitylene-derived backbones replacing\nthe trilactone core of Ent, respectively, and their monoglucosylated\ncongeners as substrates of IroB. Time course analyses and steady-state\nkinetic studies, which were performed under conditions that provide\nenhanced activity relative to prior studies, inform the substrate\nselectivity and catalytic efficiencies of this enzyme. We extend these\nfindings to the preparation of a siderophore–antibiotic conjugate\ncomposed of monoglucosylated TC and ampicillin (MGT-Amp). Examination\nof its antibacterial activity and receptor specificity demonstrates\nthat MGT-Amp targets pathogenicity because it shows specificty for\nthe pathogen-associated outer membrane receptor IroN. Overall, our\nfindings extend the biochemical characterization of IroB and its substrate\nscope and illustrate the ability to leverage a bacterial <i>C</i>-glucosyltransferase for non-native chemoenzymatic transformations\nalong with potential applications of salmochelin mimics.