Re-evaluation of the <i>C</i>-Glucosyltransferase IroB Illuminates Its Ability to <i>C</i>-Glucosylate Non-native Triscatecholate Enterobactin Mimics

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