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

Abstract

The pathogen-associated C-glucosyltransferase IroB is involved in the biosynthesis of salmochelins, C-glucosylated derivatives of enterobactin (Ent), which is a triscatecholate siderophore of enteric bacteria including Salmonella enterica and Escherichia coli. Here, we reassess the ability of IroB to C-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 C-glucosyltransferase for non-native chemoenzymatic transformations along with potential applications of salmochelin mimics.

Figure 1.

Structures of C-glucosylated Ent derivatives and Ent mimics. (a) Structures of enterobactin, MGE, DGE (Salmochelin S4), and TGE; a depiction of the general structure of class IIb microcins, showing the peptide C-terminal serine linkage to the linear MGE moiety; additional salmochelins isolated from bacterial culture, S2, S1, and SX. (b) Structures of Ent, TRENCAM, and MECAM.

Figure 2.

IroB catalyzes the successive C-glucosylation of Ent. (a) Schematic representation of the IroB-catalyzed C-glucosylation of Ent → MGE → DGE → TGE. (b) Analytical HPLC traces monitored at 220 nm of the reaction of IroB (1 μM) incubated with Ent (100 μM) and UDP-Glc (3 mM) in buffer (75 mM Tris-HCl pH 9.0, 5 mM MgCl₂, 2.5 mM TCEP) for the indicated time.

Figure 3.

IroB catalyzes the successive C-glucosylation of TC at the C5 position. (a) Schematic representation of the IroB-catalyzed C-glucosylation of TC → MGT → DGT. (b) Analytical HPLC traces monitored at 220 nm of the reaction of IroB (1 μM) incubated with TC (100 μM) and UDP-Glc (3 mM) in buffer (75 mM Tris-HCl pH 9.0, 5 mM MgCl₂, 2.5 mM TCEP) for the indicated time. (c) Selected portions of the ¹H-¹³C HMBC spectrum of MGT showing ³J couplings between C1’ and H4/H6, ²J coupling between C5 and H1’, ³J couplings between C4/C6 and H1’, and a ³J coupling between C5 and H2’, with a numbering scheme for the glucose and DHB moieties.

Figure 4.

IroB catalyzes the successive C-glucosylation of MC at the C5 position. (a) Schematic representation of the IroB-catalyzed C-glucosylation of MC → MGM → DGM. (b) Analytical HPLC traces monitored at 220 nm of the reaction of IroB (1 μM) incubated with MC (100 μM) and UDP-Glc (3 mM) in buffer (75 mM Tris-HCl pH 9.0, 5 mM MgCl₂, 2.5 mM TCEP) for the indicated time. (c) Selected portions of the ¹H-¹³C HMBC spectrum of MGM showing ³J couplings between C1’ and H4/H6, ²J coupling between C5 and H1’, ³J couplings between C4/C6 and H1’, and a ³J coupling between C5 and H2’, with a numbering scheme for the glucose and DHB moieties.

Figure 5.

Kinetic traces for IroB-catalyzed C-glucosylation of Ent, TC, MC, and their derivatives. Determined for 100 nM IroB in the presence of 500 μM UDP-Glc in buffer (75 mM Tris-HCl pH 9.0, 5 mM MgCl₂, 2.5 mM TCEP), n = 3, mean ± std.

Figure 6.

IroB catalyzes the C-glucosylation of TC-PEG₃-N_3. (a) Schematic representation of the IroB-catalyzed C-glucosylation of TC-PEG₃-N₃ → MGT-PEG₃-N₃ → DGT-PEG₃-N₃. (b) Analytical HPLC traces monitored at 220 nm of the reaction of IroB (1 μM) incubated with TC-PEG₃-N₃ (100 μM) and UDP-Glc (3 mM) in buffer (75 mM Tris-HCl pH 9.0, 5 mM MgCl₂, 2.5 mM DTT) for the indicated time.

Figure 7.

MGT-Amp exhibits antibacterial activity against STm. (a) Structure of MGT-Amp. (b) Antibacterial activity of MGT-Amp compared to that of TC-Amp and Amp. Assays conducted in modified M9 medium, 20 h, 30 °C, n = 3, mean ± std; see Figure S22a for results from assay conducted in 50% MHB + 100 μM DP.

Figure 8.

MGT-Amp antibacterial activity against STm requires the presence of OM receptor IroN. (a) Antibacterial activity of MGT-Amp against STm and its mutants in fepA, iroN, and fepA iroN; see Figure S22 for results from assay conducted in 50% MHB + 100 μM DP. Bacterial growth monitored by (b) OD₆₀₀ and (c) CFU counting for cultures of STm iroN only, 1:1 STm iroN / STm fepA coculture, and STm fepA only untreated, treated with 0.2 μM TC-Amp, or treated with 0.2 μM MGT-Amp; * indicates no colony formation. Assays were conducted in modified M9 medium, 20 h, 30 °C, n ≥ 3, mean ± std.

Figure 9.

MGT-Amp shows attenuated antibacterial activity against nonpathogenic Ec K12 and selectively kills STm in the presence of Ec K12. (a) Antibacterial activity of MGT-Amp, TC-Amp, and Amp against Ec K12. (b) Bacterial growth monitored by OD₆₀₀ for cultures of STm only, 1:1 STm/Ec K12 coculture, and Ec K12 only untreated or treated with 1 μM MGT-Amp. (c) Representative images of colonies from cultures of STm only, 1:1 STm/Ec K12 coculture, and Ec K12 only untreated or treated with 1 μM MGT-Amp plated on MacConkey agar; arrows indicate different colored colonies visible in an enlarged image of the coculture; no colonies formed for STm only treated with MGT-Amp. Assays were conducted in 50% MHB + 100 μM DP, 20 h, 30 °C, n = 3, mean ± std.