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. 2009 Sep 9;131(35):12682-92.
doi: 10.1021/ja903051q.

Enzymatic hydrolysis of trilactone siderophores: where chiral recognition occurs in enterobactin and bacillibactin iron transport

Affiliations

Enzymatic hydrolysis of trilactone siderophores: where chiral recognition occurs in enterobactin and bacillibactin iron transport

Rebecca J Abergel et al. J Am Chem Soc. .

Abstract

Bacillibactin and enterobactin are hexadentate catecholate siderophores produced by bacteria upon iron limitation to scavenge ferric ion and seem to be the ultimate siderophores of their two respective domains: Gram-positive and Gram-negative. Iron acquisition mediated by these trilactone-based ligands necessitates enzymatic hydrolysis of the scaffold for successful intracellular iron delivery. The esterases BesA and Fes hydrolyze bacillibactin and enterobactin, respectively, as well as the corresponding iron complexes. Bacillibactin binds iron through three 2,3-catecholamide moieties linked to a trithreonine scaffold via glycine spacers, whereas in enterobactin the iron-binding moieties are directly attached to a tri-l-serine backbone; although apparently minor, these structural differences result in markedly different iron coordination properties and iron transport behavior. Comparison of the solution thermodynamic and circular dichroism properties of bacillibactin, enterobactin and the synthetic analogs d-enterobactin, SERGlyCAM and d-SERGlyCAM has determined the role of each different feature in the siderophores' molecular structures in ferric complex stability and metal chirality. While opposite metal chiralities in the different complexes did not affect transport and incorporation in Bacillus subtilis, ferric complexes formed with the various siderophores did not systematically promote growth of the bacteria. The bacillibactin esterase BesA is less specific than the enterobactin esterase Fes; BesA can hydrolyze the trilactones of both siderophores, while only the tri-l-serine trilactone is a substrate of Fes. Both enzymes are stereospecific and cannot cleave tri-d-serine lactones. These data provide a complete picture of the microbial iron transport mediated by these two siderophores, from initial recognition and transport to intracellular iron release.

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Figures

Figure 1
Figure 1
Molecular structures and abbreviations of the natural (top) and synthetic (bottom) siderophores discussed in this study. The iron-coordinating oxygen atoms are indicated in red.
Figure 2
Figure 2
Spectrophotometric competition titration of SGC against EDTA ([Fe3+] = 0.09 mM, [SGC] = 0.10 mM, [EDTA] = 1.0 mM, pH from 5 to 7.5, 48 h, 0.1 M KCl, 25 °C, 1 cm cell).
Figure 3
Figure 3
Circular dichroism spectra of the studied ferric complexes ([FeL] = 0.1 mM, pH 7.4, 1 cm path, 25 °C).
Figure 4
Figure 4
Iron transport mediated by [55FeIII(BB)]3-, [55FeIII(SGC)]3-, and [55FeIII(d-SGC)]3- in B. subtilis at 37 °C in iron-limited medium. Data presented are the average of three independent experiments for BB and SGC, and two independent experiments for d-SGC.
Figure 5
Figure 5
Fluorescence quenching analyses of the substrate-binding receptor protein FeuA-His6 with the five studied ferric complexes at pH 7.4. Symbols give the fluorescence data at 340 nm, and lines give the non-linear least squares calculated fits.
Figure 6
Figure 6
Reaction time courses of Bes-catalyzed hydrolysis of BB (top), Ent (middle left) and SGC (bottom left). The presence of Bes in solution did not affect d-Ent (middle right) and d-SGC (bottom right). Reaction aliquots were quenched at different time points and analyzed by HPLC. The assignment of the hydrolysis products is based on the corresponding mass spectrometry data (Figures S4-S6), and the schematic representations of the hydrolysis products (cyclic trimer, linear trimer, dimer and monomer) are shown consistently with similar previous studies.
Figure 7
Figure 7
Bes-catalyzed hydrolysis of [FeIII(BB)]3- to [FeIII(2,3-DHBGT)3]3- followed by UV-vis spectroscopy ([Fe(BB)] = 100 μM, [Bes] = 1 μM, 75mM HEPES, pH 7.5, 25 °C, 1 cm cell). The inset shows the spectra at t = 0 h (blue) and t = 12 h (pink).

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