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

Article abstract of DOI:10.1021/ja903051q

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 acquis

Full text of DOI:10.1021/ja903051q

Published on Web 08/11/2009
Enzymatic Hydrolysis of Trilactone Siderophores: Where
Chiral Recognition Occurs in Enterobactin and Bacillibactin
Iron Transport1
Rebecca J. Abergel, Anna M. Zawadzka, Trisha M. Hoette, and
Kenneth N. Raymond*
Department of Chemistry, UniVersity of California, Berkeley, California 94720-1460
Received April 28, 2009; E-mail: raymond@socrates.berkeley.edu
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.
mediated by the energy-transducing TonB-ExbB-ExbD system.3,6
Periplasmic binding proteins shuttle the complexes from the
Introduction
The chemical activity of available ferric ion is limited to 10-18
M by the low solubility of ferric hydroxide in aerobic conditions
at physiological pH,2 and yet a 10-6 M internal iron concentra-
tion is required by bacteria to achieve optimal growth.3 To
overcome this discrepancy, a central microbial iron acquisition
mechanism is the production of low molecular weight iron-
chelating agents, called siderophores.4 Remarkable in their high
specificity and affinity toward ferric ion, siderophores are
exported from the cell in their apo form and can remove iron
from minerals and host iron-binding proteins to form stable ferric
complexes.5 Gram-negative bacteria incorporate ferric-sidero-
phore complexes via specific outer membrane receptors in a
process driven by the cytoplasmic membrane potential and
outer membrane receptors to cytoplasmic membrane ATP-
binding cassette (ABC) transporters that, in turn, deliver the
ferric-siderophores to the cytoplasm.7 Gram-positive bacteria,
which lack the lipopolysaccharide layer encasing the cell, require
only a lipoprotein tethered to the external surface of the
cytoplasmic membrane and binding-protein-dependent ABC
permeases to transport their siderophores into the cell.8 Bacteria
often possess multiple membrane receptors, each providing the
organism with specificity for different siderophores; it is also
typical for bacterial strains to utilize xenobiotic siderophores,
suggesting that the recognition process is common throughout
the microbial world.9
Enterobactin (Ent) and bacillibactin (BB) are two remarkable
hexadentate iron chelators:10 both ligands coordinate iron
through three 2,3-catecholate units that are amide linked to a
trilactone macrocycle (Figure 1); they have the highest known
(1) Paper 86 in the series “Microbial Iron Tranport Compounds.” For
previous paper, see: Zawadzka, A. M.; Abergel, R. J.; Nichiporuk,
R.; Andersen, U. N.; Raymond, K. N. Biochemistry 2009, 48, 3645–
3657.
(2) Harris, W. R. In Molecular and Cellular Iron Transport; Templeton,
D. M., Ed.; Marcel Dekker, Inc.: New York, 2002; pp 1-40.
(3) Braun, V.; Hantke, K.; Koster, W. In Metal ions in biological systems;
Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 1998; Vol. 35,
pp 67-145.
(6) Andrews, S. C.; Robinson, A. K.; Rodriguez-Quinones, F. FEMS
Microbiol. ReV. 2003, 27, 215–237.
(7) Winkelmann, G. Microbial Transport Systems; Wiley-VCH: Wein-
heim, 2001.
(4) Raymond, K. N.; Dertz, E. A. In Iron transport in bacteria; Crosa,
J. H., Mey, A. R., Payne, S. M., Eds.; ASM Press: Washington, D.C.,
2004, pp 3-17.
(8) Brown, J. S.; Holden, D. W. Micr. Infec. 2002, 4, 1149–1156.
(9) Weinberg, E. D. BioMetals 2000, 13, 85–89.
(10) Raymond, K. N.; Dertz, E. A.; Kim, S. S. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 3584–3588.
(5) Boukhalfa, H.; Crumbliss, A. L. BioMetals 2002, 15, 325–339.
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10.1021/ja903051q CCC: $40.75  2009 American Chemical Society

Chiral Recognition of Siderophores by Esterases
A R T I C L E S
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.
ferric complex formation constants (1049 and 1048, respectively),
yet determined for siderophores.11,12 First isolated in 1970 from
Escherichia coli13 and Salmonella typhimurium,14 the triserine-
based Ent is produced by a large number of enteric bacteria
and was, for almost 30 years, the only example of a siderophore
incorporating a trilactone. In contrast, the trithreonine-based BB
was recently isolated from Bacillus subtilis,15 Bacillus anthracis,
Bacillus cereus and Bacillus thuringiensis,16-18 its hydrolyzed
version was found in Bacillus licheniformis19 and genes
encoding its production were reported in Bacillus amylolique-
faciens,20 indicating that this siderophore may be the counterpart
of Ent in Gram-positive bacteria.
The extremely high affinity for iron displayed by Ent and
BB originates from the presence of the 2,3-dihydroxybenzamide
motifs. However, incorporation of these powerful 2,3-catecholate
iron-binding units in Ent and BB also makes the siderophores
and their respective ferric complexes specific targets of the
mammalian protein Siderocalin, a component of the antibacterial
iron-depletion defense of the innate immune system.21,22 Sid-
erocalin acts as a growth inhibitor of pathogens that rely solely
on Ent-mediated iron acquisition and is speculated to have the
same bacteriostatic effect on BB-dependent strains.22,23 The
siderophore recognition patterns by Siderocalin or bacterial
cognate proteins are likely different and are a determining factor
of bacterial survival and infection. Understanding the processes
involved in Ent- and BB-mediated bacterial iron acquisition is
therefore important to further explore the relationship between
siderophore production and pathogenicity. One strategy used
by pathogenic Ent-producing strains, such as E. coli, Salmonella
spp., and Klebsiella pneumoniae, is to evade siderophore
sequestration by Siderocalin Via the production of C-glucosy-
lated Ent analogs, called salmochelins.24-26 To date, Ent, BB
and the triserine-based salmochelin S4 (diglucosyl enterobactin,
DGE, Figure 1) are the only trilactone siderophores isolated
(11) Loomis, L. D.; Raymond, K. N. Inorg. Chem. 1991, 30, 906–911.
(12) Dertz, E. A.; Xu, J.; Stintzi, A.; Raymond, K. N. J. Am. Chem. Soc.
2006, 128, 22–23.
(13) O’Brien, I. G.; Gibson, F. Biochim. Biophys. Acta 1970, 215, 393–
397.
(14) Pollack, J. R.; Neilands, J. B. Biochem. Biophys. Res. Commun. 1970,
38, 989–992.
(15) May, J. J.; Wendrich, T. M.; Marahiel, M. A. J. Biol. Chem. 2001,
276, 7209–7217.
(16) Koppisch, A. T.; Browder, C. C.; Moe, A. M.; Shelley, J. T.; Kinkel,
B. A.; Hersman, L. E.; Iyer, S.; Ruggiero, C. E. BioMetals 2005, 18,
577–585.
(17) Koppisch, A. T.; Dhungana, S.; Hill, K. K.; Boukhalfa, H.; Heine,
H. S.; Colip, L. A.; Romero, R. B.; Shou, Y.; Ticknor, L. O.; Marrone,
B. L.; Hersman, L. E.; Iyer, S.; Ruggiero, C. E. BioMetals 2008, 21,
epub.
(21) Goetz, D. H.; Holmes, M. A.; Borregaard, N.; Bluhm, M. E.; Raymond,
K. N.; Strong, R. K. Mol. Cell 2002, 10, 1033–1043.
(22) Abergel, R. J.; Wilson, M. K.; Arceneaux, J. E. L.; Hoette, T. M.;
Strong, R. K.; Byers, B. R.; Raymond, K. N. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 18499–18503.
(18) Wilson, M. K.; Abergel, R. J.; Raymond, K. N.; Arceneaux, J. E. L.;
Byers, B. R. Biochem. Biophys. Res. Commun. 2006, 348, 320–325.
(19) Temirov, Y. V.; Esikova, T. Z.; Kashparov, I. A.; Balshova, T. A.;
Vinokurov, L. M.; Alakhov, Y. B. Russ. J. Bioorg. Chem. (Engl.
Transl.) 2003, 29, 542–549.
(23) Flo, T. H.; Smith, K. D.; Sato, S.; Rodriguez, D. J.; Holmes, M. A.;
Strong, R. K.; Akira, S.; Aderem, A. Nature 2004, 432, 917–921.
(24) Bister, B.; Bischoff, D.; Nicholson, G.; Valdebenito, M.; Schneider,
K.; Winkelmann, G.; Hantke, K.; Sussmuth, R. D. BioMetals 2004,
17, 471–481.
(20) Chen, X.; Koumoutsi, A.; Scholz, R.; Borriss, R. J. Mol. Microbiol.
Biotechnol. 2009, 16, 14–24.
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A R T I C L E S
Abergel et al.
from bacterial cultures and, due to their structural differences,
all three siderophores require multiple, but partially overlapping,
pathways for iron incorporation, as shown in uptake studies
performed on S. typhimurium and B. subtilis.12,27 Nevertheless,
a particular common pathway is taken by Ent, BB and DGE,
once their ferric complexes are incorporated in the cytoplasm:
in contrast to most ferric-siderophores that undergo reduction
followed by spontaneous release or competitive sequestration of
the reduced species,28 intracellular iron release from [FeIII(Ent)]3-,
[FeIII(BB)]3- and [FeIII(DGE)]3- requires hydrolysis of the trilactone
scaffolds by the specific esterases Fes, BesA and IroD, respect-
ively.29,30 Cleavage of the ester functionalities of the siderophore
backbone results in a dramatic loss of complex stability and
corresponding increase in reduction potential,31 which facilitates
subsequent removal of iron by other cellular iron-binding
components.
Despite the close similarity of Ent- and BB-mediated iron
uptake pathways, there are significant differences in the
complexes’ structures. What is the effect of the different
molecular structures of these siderophores on the specific
recognition of the ligands and their ferric complexes by
biological receptors and enzymes? Especially, how does the
incorporation of particular chiral amino acids in the trilactone
scaffolds affect the iron-coordination properties of the chelators
and the bacterial uptake and use of their respective ferric
complexes? It has long been known that Ent effectively delivers
iron for growth whereas its synthetic mirror image does not,32
yet both are recognized by the outer membrane receptor FepA.33
So where does chiral recognition occur?
metrical, hexadentate catecholate ligands; however, while the
iron-binding motifs are directly attached to a triserine scaffold
through amide linkages in Ent, BB is formed from a trithreonine
skeleton connected to the catecholamide subunits via glycine
spacers. Though the wide range of Ent- or BB-producing
microorganisms synthesize one or the other siderophore, some,
such as Salmonella enterica serovar typhimurium34 and B.
subtilis,12,35 can incorporate and use both in parallel for iron
acquisition. By affecting the iron coordination chemistry proper-
ties of the chelators, the different structures of Ent and BB are
responsible for the discrimination between their ferric complexes
by bacterial strains. The coordination chemistry of Ent, BB and
synthetic analogs, as well as their iron transport and release
properties in selected organisms, are explored here to correlate
the chemical structures of the siderophores to their biological
recognition.
Thermodynamic Stability and Induced Chirality of Trilactone-
Based Ferric Siderophore Complexes. The structural changes
between Ent and BB (Figure 1) result in these two siderophores
having different affinities for iron.12,36 The trilactone backbone
of Ent appears perfect for the size of the ferric ion, and addition
of the glycine spacer seems detrimental to the overall stability,
as suggested by the lowered thermodynamic stability of ferric
BB compared to ferric Ent.12 However, another important
change is the addition of methyl groups on the BB trilactone.
The conformation of the 12 membered rings in Ent and BB
can be viewed as three fused cyclohexane-like rings in which
the central atom is missing. There are two solutions to the
requirement that each ring must be in a chair conformation:
one in which the amide functionalities are in axial positions
([FeIII(Ent)]3-, Figure S1, Supporting Information) and one
where they are in equatorial positions ([FeIII(BB)]3-, Figure S1,
Supporting Information).37 In order to confirm that the ther-
modynamic behavior of BB is dominated by the pres-
ence of the glycine spacers and not by the incorporation of
threonine units, thermodynamic measurements were performed
on the intermediate synthetic analog: SERglyCAM (SGC, also
known as serine-corynebactin and serine-bacillibactin, Figure
1).36,37 This ligand incorporates three 2,3-catecholamide moieties
linked to a triserine lactone through glycine spacers; its synthesis
has been described earlier and it was prepared similarly.37
Formation of a ferric siderophore complex requires deprotona-
tion of the catecholate phenol groups, so determination of ligand
pKa values is crucial to evaluating the stability of ferric
complexes. Ligand protonation constants were determined for
SGC and the ferric formation and protonation constants were
evaluated. Previous studies of Ent and its analogs established
the protonation constants (pKa1-pKa3) of the meta-hydroxyl
oxygen atoms are well separated from the ortho-hydroxyl
oxygen atoms (pKa4-pKa6).11 Thus, three of the six stepwise
protonation constants (K0ln, for n ) 1-6), as defined by the
equations (1) and (2), were determined potentiometrically.
To address these questions, we report solution thermody-
namic, circular dichroism and receptor-binding studies on
synthetic analogs of Ent and BB as well as bacterial uptake
experiments and growth assays performed with B. subtilis
strains. We also describe the specificity of the esterases BesA
and Fes in the enzymatic hydrolysis of different trilactone
backbones. The results presented herein demonstrate how
molecular recognition is achieved by bacterial species through
subtle changes in the structures of the two most powerful
siderophores.
Results and Discussion
Enterobactin and Bacillibactin: Two Siderophore Archetypes.
The architectures of Ent and BB make these siderophores
predisposed to ferric ion binding and formation of highly stable
octahedral complexes.10 Both iron chelators are 3-fold sym-
(25) Fischbach, M. A.; Lin, H.; Zhou, L.; Yu, Y.; Abergel, R. J.; Liu, D. R.;
Raymond, K. N.; Wanner, B. L.; Strong, R. K.; Walsh, C. T.; Aderem,
A.; Smith, K. D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16502–
16507.
(26) Fischbach, M. A.; Lin, H. N.; Liu, D. R.; Walsh, C. T. Nature Chem.
Biol. 2006, 2, 132–138.
(27) Rabsch, W.; Methner, U.; Voigt, W.; Tschape, H.; Reissbrodt, R.;
Williams, P. H. Infect. Immun. 2003, 71, 6953–6961.
(28) Miethke, M.; Marahiel, M. A. Microbiol. Mol. Biol. ReV. 2007, 71,
413–451.
(29) Lin, H.; Fischbach, M. A.; Liu, D. R.; Walsh, C. T. J. Am. Chem.
Soc. 2005, 127, 11075–11084.
(30) Miethke, M.; Klotz, O.; Linne, U.; May, J. J.; Beckering, C. L.;
Marahiel, M. A. Mol. Microbiol. 2006, 61, 1413–1427.
(31) Cooper, S. R.; McArdle, J. V.; Raymond, K. N. Proc. Natl. Acad.
Sci. U.S.A. 1978, 75, 3551–3554.
(34) Rabsch, W.; Voigt, W.; Reissbrodt, R.; Tsolis, R. M.; Baumler, A. J.
J. Bacteriol. 1999, 181, 3610–3612.
(35) Ollinger, J.; Song, K.; Antelmann, H.; Hecker, M.; Helmann, J. D. J.
Bacteriol. 2006, 188, 3664–3673.
(32) Neilands, J. B.; Erickson, T. J.; Rastetter, W. H. J. Biol. Chem. 1981,
256, 3831–3832.
(36) Bluhm, M. E.; Kim, S. S.; Dertz, E. A.; Raymond, K. N. J. Am. Chem.
Soc. 2002, 124, 2436–2437.
(33) Thulasiraman, P.; Newton, S. M.; Xu, J.; Raymond, K. N.; Mai, C.;
Hall, A.; Montague, M. A.; Klebba, P. E. J. Bacteriol. 1998, 180,
6689–6696.
(37) Bluhm, M. E.; Hay, B. P.; Kim, S. S.; Dertz, E. A.; Raymond, K. N.
Inorg. Chem. 2002, 41, 5475–5478.
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Chiral Recognition of Siderophores by Esterases
A R T I C L E S
Table 1. Solution Thermodynamic Parameters for the
[MmLlHh]
[M]m[L]l[H+]h
mM + lL + hH+ T MmLlHh ꢀmlh
)
Trilactone-Based Ent, BB and SGC
SERglyCAM
enterobactina
bacillibactinb
(1)
(2)
[L]6-
[LH]
log K1
log K2
log K3
log K4
log K5
log K6
Σlog K1-6
log ꢀ110
12.1
12.1
12.1
8.37(3)
7.54(2)
6.68(3)
58.89
44.1(2)
29.6
12.1
12.1
12.1
8.6
7.5
6.0
58.4
49
34.3
12.1
12.1
12.1
8.43
7.43
6.77
58.93
47.6
33.1
5-
[HnL]
ꢀ0ln
ꢀ0l(n-1)
[LH2]4-
[LH3]3-
[LH4]2-
[LH5]-
K0ln
)
)
[Hn-1L][H+]
The stability constant (eq 1) of the SGC ferric complex was
measured via a competition experiment with ethylene-diamine
tetraacetic acid (EDTA), by monitoring the changes in UV-vis
absorbance (Table 1). This spectrophotometric titration exploits
the broad ligand-to-metal-charge-transfer (LMCT) band which
increases in intensity as iron exchanges from the colorless EDTA
complex to the red [FeIII(SGC)]3- complex (Figure 2). The
protonation constants obtained for SGC (Table 1) confirm that
the insertion of a glycine spacer between the trilactone backbone
and the catecholamide arms slightly alters the acidity of the
ligand when compared to Ent. The addition of the amino acid
spacer is also detrimental to the overall thermodynamic stability
of the iron complex. However, methylation of the trilactone in
BB provides a more stable iron complex than the ferric SGC.
The calculated pM values of all three chelators Ent,11 BB12 and
SGC (Table 1) corroborate that insertion of a threonine trilactone
optimizes the iron-coordination in a structure containing glycinyl
arms but does not compensate for the loss of iron-complex
stability in comparison to the more compact Ent molecule. (The
pM value is a direct measure of the chemical free energy of the
metal ion at equilibrium with the chelating siderophore. It is
defined as the negative logarithm of the free iron concentration
in equilibrium with complexed and free ligand, at physiological
pH (pH 7.4) with 1 µM total iron concentration and 10 µM
total ligand concentration.) Similar studies on an additional
analog containing three catecholamide units directly linked to
a trithreonine backbone are necessary to complete this analysis
and assess the importance of glycine insertion in the BB
structure. Unfortunately and despite several individual efforts
(Ramirez et al.38 and unpublished results), the synthesis of the
threonine trilactone precursor has not been performed success-
fully yet.
[FeL]3-
pM(FeIII
)
a Parameters determined in previous studies.11 b Parameters
determined in previous studies.12
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).
A notable feature of metal complexes formed with these
trilactone-based siderophores is their distinct chirality at the
metal center, as evidenced by circular dichroism in previous
studies.39 All three complexes [FeIII(Ent)]3-, [FeIII(BB)]3- and
[FeIII(SGC)]3- exhibit intense high energy CD bands (<390 nm)
corresponding to ligand centered π-π* transitions, as well as
characteristic ferric catechol transitions in the visible region.
The latter arise from LMCT transitions and are sensitive to the
chirality at the metal center. While the absolute configuration
of [FeIII(Ent)]3- was established as ∆,40 both ferric complexes
of SGC and BB adopt the opposite Λ chirality,39 similar to
D-enterobactin (D-Ent, enantio-enterobactin), the synthetic enan-
tiomer of Ent composed of a D-serine trilactone, which perforce
forms a Λ ferric complex. To provide a complete set of
compounds to the following study, D-SERglyCAM (D-SGC),
the enantiomer of SGC incorporating a tri-D-serine lactone, has
been prepared following the same synthetic procedure as for
SGC: coupling of the trihydrochloride salt of the D-serine
Figure 3. Circular dichroism spectra of the studied ferric complexes ([FeL]
) 0.1 mM, pH 7.4, 1 cm path, 25 °C).
trilactone to three equivalents of 2,3-bis(benzyloxy)-glyci-
nylfluoride benzoylamide, and subsequent hydrogenation with
a Pd/C catalyst afforded the enantiomerically pure hexadentate
ligand D-SGC (Scheme S1, Supporting Information).
The ferric complexes [FeIII(SGC)]3- and [FeIII(D-SGC)]3-
display opposite chiralities, as shown by circular dichroism
(Figure 3, Table 2). Incorporation of chiral amino acids in the
trilactone scaffold of siderophores such as Ent and BB induces
the chirality of the corresponding ferric complexes. However,
insertion of glycine spacers on each catecholate arms of BB or
SGC inverts the ferric complex configuration as compared to
Ent. In contrast, methylation of the trilactone backbone alone
(38) Ramirez, R. J. A.; Karamanukyan, L.; Ortiz, S.; Gutierrez, C. G.
Tetrahed. Lett. 1997, 38, 749–752.
(39) Dertz, E. A.; Xu, J.; Raymond, K. N. Inorg. Chem. 2006, 45, 5465–
5478.
(40) Karpishin, T. B.; Dewey, T. M.; Raymond, K. N. J. Am. Chem. Soc.
1993, 115, 1842–1851.
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A R T I C L E S
Abergel et al.
Table 2. Chiral Features of the Ligand and Ferric Complexes
Studied
chirality
trilactone
π-π* transitions
∆ꢀ
LMCT transitions
λmax ∆ꢀ
metal center
)
ligand
Ent
D-Ent
SGC
D-SGC
BB
amino acid
([FeIII(L)]3-
λmax (nm)
(M-1 cm-1
)
(nm) (M-1 cm-1
)
L
∆
Λ
Λ
∆
Λ
326
325
+348
-345
528
530
-133
+132
+58
D
L
310, 353 -126, +192 548
313, 352 +101, -180 548
311, 354 -137, +142 545
D
-58
a
+65
L or L-allo
a The configuration of the threonine second carbon stereocenter
(numbered 3) has not been established.
does not affect the metal center chirality. Comparison of the
π-π* transitions in [FeIII(BB)]3- and [FeIII(SGC)]3- may also
provide some information about the chirality of the trithreonine
lactone. The identical splitting of the high energy CD bands
(in two bands with peaks at 311 and 354 nm) for both complexes
is indicative of similar S chiral configuration adjacent to the
amide functionalities linking the trilactone to the arms.41 This
conformation is consistent with scaffolds formed with L-serine
or L-threonine. Nevertheless, the dimodular nonribosomal pep-
tide synthetase DhbF adenylates threonine during the biosyn-
thesis of BB in B. subtilis and has selectivity not only for the
predicted substrate L-threonine (2S, 3R) but also for the
miscognate substrate allo-L-threonine (2S, 3S).15 Thus, BB could
be based on either L-threonine or allo-L-threonine units and the
absolute configuration of the second stereocenter in each
threonine unit remains to be established.
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.
Transport and Incorporation of Trilactone-Based Catecholate
Siderophores by B. subtilis. Both BB and its precursor Itoic acid
(DHBG, the glycine conjugate of 2,3-dihydroxybenzoic acid)
have previously been characterized as the two endogenous
siderophores of B. subtilis.15 However, due to its relatively weak
affinity for iron, DHBG is unlikely to have physiological
relevance as a siderophore under natural conditions and BB has
been established as the main extracellular ferric ion scavenger
produced by B. subtilis under iron limitation.35,42 Furthermore,
B. subtilis, like many bacteria, can utilize a range of exogenous
siderophores, such as the hydroxamates ferrichrome, ferriox-
amine, coprogen, schizokinen and arthrobactin, or the catecho-
late Ent.35,42 Recent mutational analyses and transport studies
have revealed at least two catecholate transport mechanisms are
operative in B. subtilis. The major catecholate feuABC-encoded
ABC transporter coupled to the yusV-encoded ATPase promotes
the uptake of both [FeIII(BB)]3- and [FeIII(Ent)]3-, as shown by
growth stimulation assays35 and fluorescence spectroscopy,30
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 nonlinear least-
squares calculated fits.
siderophore incorporation, the larger size and elongated shape
of [FeIII(BB)]3-, [FeIII(SGC)]3- and [FeIII(D-SGC)]3- (resulting
from the insertion of glycine spacers) are therefore more likely
to be the discriminating features of the second Ent receptor.
To confirm that all five ferric-siderophore complexes can be
incorporated through the major catecholate transporter FeuABC,
the interactions of each complex with the substrate-binding
protein FeuA were characterized using fluorescence spectros-
copy. FeuA was recombinantly produced as a C-terminal His6-
tag fusion and purified by Ni affinity chromatography, following
a modified described procedure.30 To determine the equilibrium
dissociation constant of FeuA for each ferric-siderophore
complex, aliquots of freshly prepared solutions of the complex
(6 µM) were added successively to a freshly isolated FeuA-
His6 solution (100 nM) and the fluorescence intensity of the
mixture (λexc ) 281 nm, λem ) 340 nm) was measured after 5
min of equilibration. The Kd values calculated by nonlinear least-
squares analysis of the fluorescence titration curves (Figure 5)
are reported in Table 3. As previously discussed by Miethke et
al.,30 both [FeIII(BB)]3- and [FeIII(Ent)]3- specifically bind to
FeuA, with similar affinities (Kd ) 15 ( 4 nM and 19 ( 5 nM,
respectively). Correspondingly, all three synthetic analogs
[FeIII(D-Ent)]3-, [FeIII(SGC)]3- and [FeIII(D-SGC)]3- exhibit
whereas another unidentified secondary receptor does not
12,43
recognize [FeIII(BB)]3- and transports only [FeIII(Ent)]3-
.
It has been proposed that the opposite chirality of the ferric
complexes formed with Ent and BB accounts for the discrimina-
tion between both siderophores by receptors.36,37 However, not
only does D-Ent have the same transport properties as Ent,12
both [FeIII(SGC)]3- and [FeIII(D-SGC)]3- are transported into
B. subtilis at levels similar to that of [FeIII(BB)]3-, as shown
by 55Fe-siderophore uptake experiments performed on B. subtilis
ATCC 6051 cells cultured in iron-limited medium (Figure 4).
Since chirality at the metal center does not affect ferric
(41) Karpishin, T. B.; Stack, T. D. P.; Raymond, K. N. J. Am. Chem. Soc.
1993, 115, 3052–3055.
(42) Moore, C. M.; Helmann, J. D. Curr. Opin. Microbiol. 2005, 8, 188–
195.
(43) Dertz, E. A.; Stintzi, A.; Raymond, K. N. J. Biol. Inorg. Chem. 2006,
11, 1087–1097.
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Chiral Recognition of Siderophores by Esterases
A R T I C L E S
Table 3. Kd Values Determined by Fluorescence Quenching for
the Binding of Ferric Complexes by the Receptor Protein FeuA at
pH 7.4a
Table 4. Growth Promotion of B. subtilis HB5600 (dhbA::spc) and
B. subtilis HB5625 (feuA::spc dhbA::mls) by Natural and Synthetic
Trilactone-Based Siderophoresa
FeuA substrate
Kd nM (esd’s)
growth of B. subtilis straina
(diameter of growth zone, mm)
[FeIII(Ent)]3-
19(5)b
10(2)
52(8)
32(4)
15(4)b
[FeIII(D-Ent)]3-
[FeIII(SGC)]3-
[FeIII(D-SGC)]3-
[FeIII(BB)]3-
siderophore
HB 5600
HB 5625
dhbA
feuA
-
+
+(21)
-
+(21)
-
+(20)
-
-
-
-
-
-
-
Ent
D-Ent
SGC
D-SGC
BB
a Uncertainties were determined from the standard deviation of three
independent titrations. b The discrepancy in the absolute Kd values
determined for these complexes in the present study, as compared to
those reported earlier,30 are attributed to the different analytical methods
used (least-squares analysis vs midpoint estimate). A significant decrease
in ferric-siderophore complex affinity was observed when stock
solutions of the protein were not prepared on the day of the fluorescence
experiment, which could also impair comparison between these and the
previously reported values.
a Growth promotion of B. subtilis strains was assessed on LB agar
plates supplemented with 0.5 mM 2,2′-dipyridyl. The genotypes of the
strains are shown as follows: +, wild type; -, mutant. Symbols: +, a
halo of growth around filter paper discs is observed; -, no growth.
only IroN recognizes [FeIII(BB)]3-.34 Correspondingly, [FeIII(D-
Ent)]3- is also incorporated in E. coli through FepA but does
not promote growth of the microorganism.32,33 In the ferric
complexes tested, the chiralities of the trilactone stereocenters
and of the metal centers must be distinguished. Both
[FeIII(BB)]3- and [FeIII(SGC)]3- exhibit Λ chirality at the ferric
center, the opposite configuration to that of [FeIII(Ent)]3- (∆),
and still deliver iron to the bacteria as efficiently as [FeIII(Ent)]3-.
In contrast, D-SGC forms a ∆ ferric complex but does not
stimulate growth of the bacterium. Thus there is a clear picture:
the chirality of the ligand trilactone, not that of the metal center,
is the selective feature of these complexes for successful
bacterial iron uptake.
specific quenching of the receptor protein, with equilibrium
dissociation constants between 10 nM and 52 nM, in the same
order of magnitude as those observed for the two complexes
derived from natural siderophores. Significantly, the ferric
complexes formed with the D-serine trilactone-based ligands,
[FeIII(D-Ent)]3- and [FeIII(D-SGC)]3-, showed slightly higher
affinities for FeuA than their respective L-serine counterparts.
The same trend was observed in the binding of [FeIII(Ent)]3-
and [FeIII(D-Ent)]3- to the E. coli periplasmic protein FepB,44
which transports ferric-Ent from the periplasm into the cyto-
plasm. These spectroscopic measurements confirm that there is
no significant discrimination in the recognition and binding of
the different ferric complexes of trilactone-based catecholate
siderophores by the FeuA substrate-binding protein, the entrance
point of the FeuABC transporter.
Utilization of Trilactone-Based Catecholate Siderophores by
B. subtilis. Active transport of a ferric-siderophore complex
through the bacterial membrane does not necessarily imply that
the iron is easily available to the bacteria. To probe whether all
five trilactone-based siderophores and synthetic analogs provide
iron usable by B. subtilis, growth promotion assays were
performed with B. subtilis HB5600 (dhbA::spc), a strain lacking
the biosynthetic enzymes for its own endogenous DHBG and
BB.35 Growth of this strain in the presence of the strong iron
chelator 2,2′-dipyridyl requires transport and utilization of an
exogenously provided siderophore. While the natural ligands
Ent and BB and the synthetic SGC all promoted growth of B.
subtilis HB5600, no significant zone of growth stimulation was
observed when discs infused with D-Ent and D-SGC were
overlaid on the bacterial cultures (Table 4). Furthermore,
deletion of the receptor feuA in B. subtilis HB5625 (feuA::spc
dhbA::mls) prevented growth stimulation by not only BB and
Ent, as reported by Helmann and co-workers,35 but also by SGC.
Thus, FeuABC is the major transporter used to incorporate the
ferric complexes of all five trilactone-based ligands into the
bacterial intracellular space, where discrimination between iron
transporters occurs. These growth stimulation experiments did
not however provide any evidence of the secondary Ent receptor
detected through direct uptake assays by Dertz et al.12 Similar
results have been reported for Ent-producing Gram-negative
bacteria: in S. typhimurium, both outer membrane receptors
FepA and IroN participate in Ent-mediated iron transport but
The Chiral Recognition of Trilactone-Based Siderophores
by Esterases. The use of BB for iron uptake by Bacilli species
requires enzymatic hydrolysis of the trithreonine backbone.30
The B. subtilis gene locus yuiI (recently named besA)30 encodes
the production of the esterase BesA, which was found to
hydrolyze both BB and [FeIII(BB)]3-, with a higher catalytic
efficiency observed for the ferric complex.30 The linear deriva-
tives of BB formed after cleavage of the ester bonds in the
lactone are based on 2,3-dihydroxybenzoyl-glycine-threonine
units (2,3-DHBGT). Miethke and co-workers have shown that
five BB-producing B. subtilis related species, including the
human pathogens B. anthracis and B. cereus, possess a yuiI
ortholog downstream of their feuABC(feuD) transport genes.30
The yuiI orthologs were found highly conserved among these
species and may correspondingly be essential for hydrolysis of
the BB scaffold and subsequent intracellular iron release in all
respective species. The esterase BesA was recombinantly
produced as a C-terminal His6-tag fusion and purified by Ni
affinity chromatography. Hydrolysis reactions by the protein
were carried out at pH 7.4, with 72 nM enzyme and 64 µM
siderophore, and followed by HPLC (Figure 6). The single
substrate concentrations used in this enzymatic assay did not
permit quantitative determination of the kinetic parameters but
were sufficient to describe the scope of substrates for BesA-
catalyzed hydrolysis. The three ligands BB, Ent and SGC were
hydrolyzed sequentially into the linear trimeric, dimeric and
monomeric derivatives of 2,3-DHBGT, 2,3-DHBS (2,3-dihy-
droxybenzoyl-serine) and 2,3-DHBGS (2,3-dihydroxybenzoyl-
glycine-serine) respectively. However BesA had no effect on
the tri-D-serine lactone of D-Ent and D-SGC. In addition, the
reactions between the ferric complexes formed with all five
siderophores and BesA were monitored by UV-vis spectros-
copy (Figures 7 and S2, Supporting Information). When BesA
was added to [FeIII(BB)]3-, [FeIII(Ent)]3- or [FeIII(SGC)]3-, the
(44) Sprencel, C.; Cao, Z.; Qi, Z.; Scott, D. C.; Montague, M. A.; Ivanoff,
N.; Xu, J.; Raymond, K. N.; Newton, S. M. C.; Klebba, P. E. J.
Bacteriol. 2000, 182, 5359–5364.
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A R T I C L E S
Abergel et al.
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, Supporting Information), and the schematic representations
of the hydrolysis products (cyclic trimer, linear trimer, dimer and monomer) are shown consistently with similar previous studies.29
respectively.29 No change in the absorption spectra was observed
when Bes was added to [FeIII(D-Ent)]3- or [FeIII(D-SGC)]3-
.
In Ent-producing species, the apo form of Ent and its ferric
complex are subject to enzymatic sequential hydrolysis by the
designated cytosolic esterase Fes, with the catalytic efficiency
being much higher for [FeIII(Ent)]3- than for apo-Ent.29 On the
other hand, neither apo-D-Ent nor [FeIII(D-Ent)]3- are hydrolyzed
by Fes.45 Enzymatic reactions with Fes from Shigella flexneri
were followed for the remaining siderophores: SGC was
hydrolyzed whereas D-SGC and BB did not react (Figure S3,
Supporting Information). Thus, Fes appears more specific than
BesA: while the L-serine scaffold is an essential feature for Fes
hydrolysis and intracellular iron release in enterobacteric species,
the esterase BesA used by Bacilli species can also hydrolyze
the threonine scaffold of BB.
Ent- and BB-Mediated Iron Transport. As known to date,
microorganisms producing Ent (or BB, respectively) do not
synthesize BB (or Ent, respectively). However, bacteria com-
monly develop several membrane receptors to appropriate
Figure 7. Bes-catalyzed hydrolysis of [FeIII(BB)]3- to [FeIII(2,3-DH-
BGT)3]3- followed by UV-vis spectroscopy ([Fe(BB)] ) 100 µM, [Bes]
) 1 µM, 75 mM HEPES, pH 7.5, 25 °C, 1 cm cell). The inset shows the
spectra at t ) 0 h (blue) and t ) 12 h (pink).
UV-vis spectra changed, as expected, to the characteristic
(45) Kim, Y.; Maltseva, N. I.; Abergel, R. J.; Binkowski, T. A.; Quartey,
P.; Dementieva, I.; Holzle, D.; Collart, F.; Raymond, K. N.; Joachim-
iak, A. Submitted for publication, 2009.
spectra of the corresponding tris-bidentate complexes [FeIII(2,3-
DHBGT)]3-, [FeIII(2,3-DHBS)3]3- and [FeIII(2,3-DHBGS)3]3-
,
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Chiral Recognition of Siderophores by Esterases
A R T I C L E S
xenobiotic siderophores for their own use. As mentioned above,
some Ent-producing bacterial strains, such as S. typhimurium,
possess two receptor proteins, FepA and IroN, for catecholate
siderophores and can transport both [FeIII(Ent)]3- and
[FeIII(BB)]3-.34 Likewise, B. subtilis can utilize Ent and acquires
Ent in E. coli. For BB, insertion of glycine spacers between
the macrocycle and the catecholate iron-binding units changes
the chirality at the metal center of the ferric complex and results
in a larger and more oblate shape; chirality at the metal center
does not affect binding by receptor proteins, whereas the shape
of the complex does. The use of a trithreonine rather than a
tri-L-serine backbone in BB is the seminal structural change
that not only confers acid resistance to the siderophore but also
prevents piracy of BB by Ent-producing bacteria. That is,
inversion of the trilactone chiral configuration does not inhibit
the transport of ferric complexes but does prevent enzymatic
hydrolysis and intracellular iron release. Ent and BB are built
on similar scaffolds and are the most powerful siderophores
used in microbial iron uptake. Nevertheless, fine-tuned changes
in the molecular structures of these ligands significantly affect
their chemical properties and subsequent recognition in different
biological systems. One consequence of understanding the
siderophore-production strategies used by various bacterial
populations in their arms race for efficient iron acquisition may
be the rational design of antibiotics based on siderophores.
its ferric complex through FeuA and a specific receptor different
12,35
from that designed for [FeIII(BB)]3-
.
Nevertheless, once
taken up, the ferric complexes must be hydrolyzed to release
iron. While BesA enzymatically cleaves both threonine and
L-serine trilactones, only the tri-L-serine macrocycle is a substrate
for Fes. Enteric strains utilizing BB must therefore encode an
ortholog of yuiI. Pathogenic E. coli and Salmonella strains that
encode the iroA gene cluster produce the esterases IroE and
IroD,29 which show approximately 37% (Basic Local Alignment
Search Tool,46,47 E ) 5 × 10-26) and 30% (E ) 2 × 10-05
)
identity to BesA, respectively, and contain the conserved
GXSXG serine esterase motif; the multiple sequence alignment
for BesA, IroE, IroD and Fes, as well as the corresponding
dendrogram are shown in Figures S7-A and S7-B, Supporting
Information. Since BB is recognized by the same S. typhimurium
receptor as DGE and both enzymes have been shown to
participate in DGE hydrolysis,29 IroE and IroD are viable
candidates for the hydrolysis of BB in yuiI-lacking bacterial
strains.
Methods
General. All chemicals were obtained from commercial suppliers
and were used as received. The starting materials tris(N-hydrochloride-
D-serine) trilactone (1)48 and 2,3-di(benzyloxy)benzoyl-glycinyl
fluoride (2),37 as well as the ligands Ent,48 D-Ent48 and SGC37 were
synthesized according to procedures described in the cited refer-
ences. The siderophore BB was isolated from B. subtilis and purified
as previously reported.18 Flash silica gel chromatography was
performed using Merck 40-70 mesh silica gel. Melting points were
taken on a Bu¨chi melting apparatus and are uncorrected. All NMR
spectra were recorded at ambient temperature on Bruker FT-NMR
spectrometers at the NMR Laboratory, College of Chemistry, UC
Berkeley. Microanalyses were performed by the Microanalytical
Services Laboratory, College of Chemistry, UC Berkeley. Mass
spectra were recorded at the Mass Spectrometry Laboratory, College
of Chemistry, UC Berkeley. UV-visible absorption spectra were
taken on a Varian Cary 300 UV-vis spectrometer. CD spectra were
recorded on a Jasco J-810 spectropolarimeter.
Significantly, proteins involved in uptake and hydrolysis of
trilactone-siderophores and their ferric complexes exhibit dif-
ferent recognition patterns: the binding of receptor proteins is
determined by the coordination chemistry around the metal
center (ferric catecholate complexes are not discriminated in
this case) whereas identity and stereochemistry of the lactone-
monomer are the selective features for backbone hydrolysis by
the corresponding esterases. It remains unknown whether the
divergent evolution and recognition of substrate-binding proteins
and hydrolases is correlated to the production of specific
siderophores. Moreover, the order of appearance of Ent and BB
as bacterial metabolites has not been determined, and it is yet
unexplained why only some BB-producing strains can produce
adequate receptors and esterases to utilize exogenous Ent, and
vice versa. It is also remarkable that, when acidic solutions (6
M HCl) of Ent and BB were stirred overnight and analyzed
(Figure S8, Supporting Information) by HPLC, Ent was
completely degraded while BB remained essentially intact; hence
the threonine macrocycle is much more resistant to acid
hydrolysis than its serine counterpart. Methylation of the Ent
backbone in BB could therefore be a bacterial strategy to
produce a siderophore resistant to acidic environment.
N,N′,N′′-Tris[2,3-di(benzyloxy)benzoyl-glycinyl]cyclotri-D-ser-
yl Trilactone, Bn6-D-SERglyCAM (3). Tris(N-hydrochloride-D-
serine) trilactone (0.176 g, 0.48 mmol) was suspended in 40 mL
of dry and degassed THF and cooled in an ice/water bath. Solu-
tions of 2,3-di(benzyloxy)benzoyl-glycinyl fluoride (2.5 mmol) in
10 mL of THF and triethylamine (0.49 g, 4.8 mmol) were added
simultaneously dropwise via syringes over 10 min into this
suspension while stirring under nitrogen. The mixture was allowed
to warm to room temperature and stirred overnight. It was then
filtered, concentrated, applied to a silica gel column and eluted with
98:2 CH2Cl2:MeOH. Fractions were combined and evaporated to
Conclusion
1
a red solid. Yield: 0.17 g (26%). H NMR (400 MHz, CDCl3): δ
While the triscatecholate ferric center found in [FeIII(Ent)]3-
and [FeIII(BB)]3- serves as the primary recognition point for
specific binding by cognate bacterial receptors or mammalian
Siderocalin, the trilactone scaffold incorporated in both sidero-
phores is the crucial feature that determines substrate recognition
by designated esterases and subsequent intracellular iron release.
This is the source of the chiral selectivity of iron delivery by
3.66 (dd, J ) 11.2 Hz, J′ ) 5.6 Hz, 3H), 3.80 (dd, J ) 11.2 Hz,
J′ ) 5.6 Hz, 3H), 4.12 (d, J ) 11.6 Hz, 3H), 4.80 (d, J ) 7.6 Hz,
3H), 4.90 (d, J ) 8.4 Hz, 3H), 5.00-5.13 (m, 12H), 7.00-7.35
(m, 36H), 7.72 (dd, J ) 3.2 Hz, J′ ) 2.8 Hz, 3H), 8.21 (d, J ) 7.6
Hz, 3H), 8.61 (br t, 3H) ppm. 13C NMR (400 MHz, CDCl3): δ
43.4, 65.0, 71.2, 117.6, 123.1, 124.5, 126.6, 127.7, 128.3, 128.4,
128.5, 128.6, 129.3, 136.3, 147.3, 152.0, 165.7, 169.0, 169.8 ppm.
Mp: 79-80 °C. (+)-FABMS: m/z 1381.5 (MH+).
N,N′,N′′-Tris[2,3-dihydroxybenzoyl-glycinyl]cyclotri-D-seryl
Trilactone, D-SERglyCAM (4). Absolute ethanol (14 mL) was
added to a suspension of Bn6-D-SERglyCAM (170 mg, 0.12 mmol)
in 100 mL of ethyl acetate. The solution was hydrogenated over
10% Pd-C (34.0 mg) at room temperature and atmospheric
hydrogen pressure for 24 h. The reaction mixture was filtered over
Celite, washed with acetone and evaporated under vacuum. The
product was collected as a light beige powder. Yield: 99.0 mg
(46) Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.;
Miller, W.; Lipman, D. J. Nucleic Acids Res. 1997, 25, 3389–3402.
(47) Altschul, S. F.; Wootton, J. C.; Gertz, E. M.; Agarwala, R.; Morgulis,
A.; Schaffer, A. A.; Yu, Y. FEBS J. 2005, 272, 5101–5109.
(48) Meyer, M.; Telford, J. R.; Cohen, S. M.; White, D. J.; Xu, J.; Raymond,
K. N. J. Am. Chem. Soc. 1997, 119, 10093–10103.
(49) Cohen, S. M.; O’Sullivan, B.; Raymond, K. N. Inorg. Chem. 2000,
39, 4339–4346.
9
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A R T I C L E S
Abergel et al.
1
(88%). H NMR (500 MHz, DMSO-d6): δ 4.01 (d, J ) 5.5 Hz,
complished using the program Hyperquad,57 which allows simul-
taneous nonlinear least-squares refinement of the data from multiple
titration curves.
6H), 4.27-4.35 (m, 6H), 4.71 (dd, J ) 8.0 Hz, J′ ) 4.5 Hz, 3H),
6.69 (t, J ) 8.0 Hz, 3H), 6.91 (d, J ) 6.5 Hz, 3H), 7.30 (d, J ) 8.0
Hz, 3H), 8.76 (d, J ) 4.5 Hz, 3H), 9.04 (s, 3H), 9.23 (s, 3H), 12.22
(s, 3H) ppm. Mp: 132-133 °C. (+)-FABMS: m/z 841 (MH+). Anal.
Calcd (Found) for C36H36N6O18.2H2O.MeOH: C, 48.90 (48.86); H,
4.88 (5.03); N, 9.25 (9.04).
Solution Thermodynamics. Protonation and ferric complex
formation constants for SGC were determined using procedures
and equipment described previously.49-51
Formation Constants: EDTA Competition Spectrophotomet-
ric Titrations. Each EDTA competition experiment consisted of a
pair of titrations: forward vs KOH and reverse vs HCl. At the lower
pH end of the titration, the colorless iron-EDTA complex domi-
nated, but as the pH was raised, the solution turned pink to red,
corresponding to the gradual formation of the iron-experimental
ligand complex. Solutions were assembled from a weighed portion
of ligand and the supporting electrolyte solution, with resulting
ligand concentrations of about 0.1 mM. The EDTA was present in
a 10-fold excess and the iron in a 0.9 fold default over the ligand
in order to prevent formation of insoluble iron species. In each
incremental experiment, an average of 24 points was collected over
a period of 48 h (∼2 h equilibration time per point). Each data
point consisted of a pH measurement and an absorbance spectrum
over at least 80 different wavelengths between 300 and 700 nm
over the pH range 5-7.5. The data were imported into the
refinement program pHab58 and analyzed by nonlinear least-squares
refinement.
Titration Solutions and Equipment. Corning high performance
combination glass electrodes (response to [H+] was calibrated before
each titration)52 were used together with either an Accumet pH
meter or a Metrohm Titrino to measure the pH of the experimental
solutions. Metrohm autoburets (Dosimat or Titrino) were used for
incremental addition of acid or base standard solutions to the
titration cell. The titration instruments were fully automated and
controlled using LabView software.53 Titrations were performed
in 0.1 M KCl supporting electrolyte under positive Ar gas pressure.
The temperature of the experimental solution was maintained at
25 °C by an external circulating water bath. UV-visible spectra
for incremental titrations were recorded on a Hewlett-Packard 8452a
spectrophotometer (diode array). Solid reagents were weighed on
a Metrohm analytical balance accurate to 0.01 mg. All titrant
solutions were prepared using distilled water that was further
purified by passing through a Millipore Milli-Q reverse osmosis
cartridge system. Titrants were degassed by boiling for 1 h while
being purged under Ar. Carbonate-free 0.1 M KOH was prepared
from Baker Dilut-It concentrate and was standardized by titrating
against potassium hydrogen phthalate using phenolphthalein as an
indicator. Solutions of 0.1 M HCl were similarly prepared and were
standardized by titrating against sodium tetraborate to Methyl Red
end point. Stock solutions of ferric ion were obtained by dissolving
solid FeCl3.6H2O in 0.1 M HCl and were standardized by titrations
with EDTA according to the methods of Welcher.54
Incremental Titrations. Initial titration solutions were assembled
from the constituent reagents in ratios determined previously by
modeling using estimated formation constants and the modeling
program Hyss.55,56 The solutions were incrementally perturbed by
the addition of either acid (HCl) or base (KOH) titrant, followed
by a time delay for equilibration (90 s for protonation studies; 2 h
for EDTA competition titrations). Potentiometric and EDTA
competition titrations were conducted in pairs: first a forward
titration from low to high pH, then a reverse titration back to low
pH. The data for the two titrations comprising each experiment
were pooled for calculation of formation constants when revers-
ibility was achieved. All absorbance measurements used for
calculation of formation constants were less than 1.05 absorbance
units.
Data Treatment. All equilibrium constants were defined as
cumulative formation constants, ꢀmlh according to eq 1, where the
n
ligand is designated as L. Stepwise protonation constants, Ka , may
be derived from these cumulative constants according to eq 2. In
the case of the competition experiments, the equilibration of iron
between SGC and EDTA was calculated by including the proton
association and iron formation constants for EDTA59 as fixed
parameters in the refinements. Because of the hexacoordinate nature
of the ligand, it was assumed that ternary (i.e., mixed EDTA-Fe-
SGC) complexes were not formed. This assumption was supported
by spectral data showing that the band shapes and λmax values of
the LMCT transitions of the Fe-SGC complex were unchanged in
the presence and absence of EDTA. Each pair of titrations (i.e.,
forward titration against KOH and reverse titration against HCl)
was combined for simultaneous refinement. For potentiometric
titrations, both the proton and ligand concentrations were refined,
only the proton concentration was allowed to vary in the spectro-
photometric studies, and all other concentrations were held at
estimated values determined from the volume of standardized stock
or the weight of ligand (measured to 0.01 mg). Refined concentra-
tions were within 5% of the analytical values. For spectral titrations,
all species formed with the ligand were considered to have
significant absorbance to be observed in the UV-vis spectra.
Circular Dichroism Spectroscopic Measurements. Solutions
for CD were made in situ. The ferric complexes were prepared
from DMSO stock solutions of the free ligands (4 mM, 25 µL)
with iron trichloride (27 mM, 3.7 µL). Sodium phosphate (pH )
7.4, 100 µL) was added to form the tris-catecholate complex, and
the solutions were diluted with water to yield a final FeL
concentration of 0.1 mM. The spectra were obtained with cuvettes
of 1 cm path length and recorded on a Jasco J-810 spectropola-
rimeter.
Protonation Constants: Potentiometric Titrations. Solutions
were assembled from a weighed portion of ligand and the supporting
electrolyte solution, with resulting ligand concentrations between
0.2 and 0.5 mM. An average of 60-90 data points were collected
in each pair of titrations (forward and back), each data point
consisting of a volume increment and a pH reading over the pH
range 4-9.5. Refinement of the protonation constants was ac-
Protein Cloning, Expression and Purification. The esterase
Fes from the human pathogen Shigella flexneri 2a str. 2457T was
purified by Dr. Natalia Maltseva (Argonne National Laboratory),
as described elsewhere.45 The substrate-binding protein FeuA from
B. subtilis 168 and the esterase BesA from B. cereus ATCC 14579
were cloned, expressed and purified as follows. The besA gene
(iroE, BC_3734) was amplified by PCR from the chromosomal
DNA using the following primers: besF (5′- CACCGTGAATAC-
TACAGTTGAAAAACAGCA-3′)andbesR(5′-TCCCTGAAAATA-
CAGGTTTTCTACGTAACTAATAAACCTCAATCCTTT-3′).These
(50) Johnson, A. R.; O’Sullivan, B.; Raymond, K. N. Inorg. Chem. 2000,
39, 2652–2660.
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6742.
(52) Gans, P.; O’Sullivan, B. Talanta 2000, 51, 33–37.
(53) Wandersman, C.; Delepaire, P. Annu. ReV. Microbiol. 2004, 58, 611–
647.
(54) Welcher, F. J. The Analytical Uses of Ethylenediamine Tetraacetic
Acid; D. van Nostrand Co.: Princeton, NJ, 1958.
(55) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A.
HYSS; Leeds: Florence, Italy, 1999.
(57) Gans, P.; Sabatini, A.; Vacca, A. HYPERQUAD; Leeds, Florence, Italy,
2000.
(58) Gans, P.; Sabatini, A.; Vacca, A. Ann. Chim. (Rome) 1999, 89, 45–
49.
(56) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A.
Coord. Chem. ReV. 1999, 184, 311–318.
(59) Smith, R. M.; Martell, A. E. Critical Stability Constants; Plenum: New
York, 1977; Vol. 1-4.
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Chiral Recognition of Siderophores by Esterases
A R T I C L E S
primers were designed to enable directional cloning into a pET101/
D-TOPO expression vector introducing a C-terminal His6-tag; a
TEV protease cleavage site was incorporated into the besR primer
to remove the tag after protein purification (underlined sequences).
The feuA gene was amplified by PCR from the chromosomal DNA
using the following primers: feuF (5′- CACCATGGGCAG-
CAAAAATGAATCAACT-3′) and feuR (5′- TCAATGGTGATG-
GTGATGATGGTTTTGTGTCAATTTTTCAGCAG-3′). The His6
sequence followed by the stop codon was introduced into the feuR
primer (underlined) so that the recombinant protein has His6 residues
added immediately at the C-terminus. The PCR products were
cloned into a pET101/D-TOPO vector using a Champion pET
Directional TOPO expression kit (Invitrogen) and used to transform
One Shot TOP10 chemically competent Escherichia coli cells. The
correct sequence of the insert was confirmed by sequencing and
the construct was transformed into chemically competent E. coli
BL21(DE3) (Invitrogen). Expression of the recombinant BesA-His6
or FeuA-His6 was induced with 0.5 mM of IPTG when the cultures
reached an OD600 of 0.6-0.8. After induction, cultures were
incubated an additional 4-5 h at 37 °C. Cells were harvested by
centrifugation at 4 °C and the pellets frozen at -20 °C. For protein
purification, the harvested cells were suspended in BugBuster
Protein Extraction Reagent Master Mix (5 mL per 1 g of wet cells)
(EMD Biosciences) and the protease inhibitor cocktail for purifica-
tion of His-tagged proteins (Sigma) was added. After 10 min of
incubation at room temperature, glycerol (5%), NaCl (300 mM)
and imidazole (10 mM) were added and the mixture was incubated
for an additional 10 min. The cell debris were removed by
centrifugation for 20 min at 16,000 × g at 4 °C. Recombinant BesA-
His6 and FeuA-His6 were purified from the clarified cell extract
using HIS-Select nickel affinity agarose gel (Sigma-Aldrich) to
>95% purity (Figure S9, Supporting Information). The equilibration
and wash buffer consisted of 50 mM sodium phosphate, pH 8.0,
0.5 M NaCl, 10% glycerol, and 10 mM imidazole, for both proteins.
The proteins were eluted with a buffer containing 50 mM sodium
phosphate, pH 8.0, 0.5 M NaCl, 10% glycerol, and 250 mM
imidazole. The purified BesA-His6 (Yield from culture of trans-
formed E. coli: 9.0 mg ·L-1) and FeuA-His6 (Yield from culture of
transformed E. coli: 2.8 mg ·L-1) were stored frozen at -20 °C in
the elution buffer. The C-terminal His6-tag was removed from the
recombinant BesA-His6 using AcTEV protease (Invitrogen). Prior
to tag removal the buffer was exchanged into Tris-buffered saline
(TBS), pH 7.4 by gel filtration (Sephadex G-25). BesA was purified
using nickel affinity chromatography. The elution buffer for BesA
and FeuA-His6 was exchanged into TBS, pH 7.4, the working buffer
for fluorescence experiments. The concentration of eluted proteins
was measured by bicinchoninic acid (BCA) protein assay. SDS-
PAGE analysis was performed on polyacrylamide gels and the gels
were stained with Coomasie Brilliant Blue R250 (Figure S9,
Supporting Information). Molecular weight and homogeneity of
BesA and FeuA-His6 were examined using electrospray ionization-
mass spectrometry.
were freshly prepared in situ. An aliquot of a DMSO stock solution
of the free ligand (4 mM, 25 µL) and FeCl3 salt (27 mM, 1 eq.,
solution standardized by EDTA titration according to the methods
of Welcher54) were combined, vigorously shaken and diluted with
TBS buffer to form the tris-catecholate complexes at a concentration
of 0.1 mM (concentrations were checked by UV-visible spectros-
copy; for [FeIII(D-SGC)]3-, ꢀ(λ ) 494 nm) ) 4900 M-1 cm-1 and
ꢀ(λ ) 338 nm) ) 15 100 M-1 cm-1). The solutions were equili-
brated for 2 h and diluted to a final concentration of 6 µM in
aqueous TBS buffer (pH 7.4).
HPLC Separation Assay. All High Pressure Liquid Chroma-
tography runs were performed in the same conditions, by injection
of 20 µL of filtered solutions (0.22 µM) through a reverse-phase
analytical C-18 column. A gradient from 5% CH3CN in ddH2O/
0.1%TFA to 50% CH3CN in ddH2O/0.1% TFA over 20 min at 1
mL/min was used to elute the siderophores and their derivatives
(detection by UV-vis absorption at 316 nm and identification by
electrospray mass spectrometry). All enzymatic reactions were
carried out in 75 mM HEPES buffer pH 7.5, as described
previously,29 with 64 µM substrates and 72 nM enzymes. Fes and
BesA frozen stocks were thawed on ice, diluted to 1 µM with cold
buffer (25 mM Tris pH 8, 50 mM NaCl, 1 mM DTT, 10% v
glycerol) and then added to the reaction mixture. The reactions were
quenched at 2, 8, 25 min and 2 h with 0.5 volumes of 2.5 N HCl
in methanol and then frozen on dry ice. For HPLC analysis, the
samples were thawed just before injection. For strong acid
hydrolysis reactions, acidic solutions of Ent and BB (1 mg/mL, 6
M HCl) were stirred for 12 h before HPLC injection.
Enzymatic Hydrolyses Followed by UV-Vis Spectroscopy.
All reactions were carried out in 75 mM HEPES buffer pH 7.5, as
described previously,29 with 100 µM substrates and 1 µM enzymes.
The ferric-siderophore complexes were prepared from DMSO stock
solutions of the free ligands (4 mM, 6.76 µL) with iron trichloride
(27 mM, 1.0 µL), and diluted to 270 µL. Fes and BesA frozen
stocks were thawed on ice, diluted to 1 µM with cold buffer (25
mM Tris pH 8, 50 mM NaCl, 1 mM DTT, 10% v glycerol) and
then added to the reaction mixture. UV-visible absorption spectra
were recorded in a 100 µL quartz cuvette (1 cm path length), over
12 h, on a Varian Cary 300 UV-vis spectrometer.
Disc Diffusion Assays. Growth assays were performed as
described in the literature61 with B. subtilis strains HB5600
(dhbA::spc) and HB5625 (feuA::spc dhbA:mls) obtained from Prof.
John Helmann (Cornell University).35 Both strains were grown to
late exponential phase in LB medium supplemented with specti-
nomycin (100 µg mL-1); erythromycin (1 µg mL-1) and linkomycin
(25 µg mL-1) were added in the case of HB5625. Cells were washed
twice with TE buffer at pH 8, resuspended in sterile water and
diluted 1:100 in 0.7% noble agar cooled to 45 °C. Aliquots of 3
mL of these suspensions were overlaid onto LB agar plates
supplemented with 0.5 mM 2,2′-dipyridyl. Sterile paper discs
infused with 20 nmoles of tested siderophore were placed on the
agar overlays, incubated at 37 °C overnight and examined for the
presence of zones of growth around the discs. The bacterial growth
can only be supported by the siderophores that have higher or
comparable affinity for iron than 2,2′-dipyridyl.
Fluorescence Quenching Binding Assay. Fluorescence quench-
ing of recombinant FeuA-His6 was measured on a Cary Eclipse
fluorescence spectrometer with 10 nm slit band-pass and a high
voltage detector, using the characteristic excitation and emission
wavelengths λexc ) 281 nm and λem ) 340 nm. Measurements were
made at a protein concentration of 100 nM in buffered aqueous
solutions, plus 32 µg/mL ubiquitin (Sigma). Fluorescence values
were corrected for dilution upon addition of substrate. Fluorescence
data were analyzed by nonlinear regression analysis of fluorescence
response versus ligand concentration using a one-site binding model
as implemented in DYNAFIT.60 The Kd values are the results of
three independent titrations. All binding experiments were done at
pH 7.4 using TBS buffer. Control experiments were performed to
ensure the stability of the protein in these experimental conditions,
and to account for dilution and photobleaching. Substrate solutions
Ferric Complex Transport Assays in B. subtilis ATCC 6051.
These assays were performed following modified described pro-
cedures and using radio-labeled ferric-siderophore complexes.43 We
chose to perform these experiments with the wild-type BB producer
B. subtilis ATCC 6051 rather than with the mutant strain B. subtilis
HB5600 (dhbA::spc) to be consistent with previous siderophore-
mediated iron uptake studies in this model organism.12,43 The iron
complexes were formed by mixing 55FeCl3 (2.8 Ci/mmol, 6.6 µL,
19 nmol) and FeCl3 (2.7 µL, 72 nmol) with the corresponding ligand
(61) Lee, J. Y.; Janes, B. K.; Passalacqua, K. D.; Pfleger, B.; Bergman,
N. H.; Liu, H.; Hakansson, K.; Somu, R. V.; Aldrich, C. C.;
Cendrowski, S.; Hanna, P. C.; Sherman, D. H. J. Bacteriol. 2007,
189, 1698–1710.
(60) Kuzmic, P. Anal. Biochem. 1996, 237, 260–273.
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A R T I C L E S
Abergel et al.
(25 µL, 100 nmol) in a ratio of 0.9:1 and ddH2O (165.7 µL). These
solutions were then incubated at room temperature for 2 h and
diluted with sodium phosphate (100 µL, 1 M, pH 7.4) and ddH2O
(700 µL). Free iron was removed by centrifugation at 14,000 rpm
for 1 min. An aliquot of each concentrated stock solution (100 µL,
91 µM Fe) was diluted with 9.9 mL of the iron-limited medium
and filtered twice through membrane filters (HAWP Millipore, 0.45
µm pore size). The concentrations of the ferric complexes in the
working solutions were quantified by determining the specific
radioactivity. B. subtilis ATCC 6051, acquired from the American
type Culture Collection and cultured on nutrient agar plates, was
grown in iron-free medium to the late exponential phase. The cells
were then washed, suspended in the iron-limited medium, and kept
on ice until the transport assay. The resuspension was set to an
OD600 of 0.61 ( 0.02. After incubation of the cells (9 mL), for 10
min at 37 °C (for cold transport assays, cells were kept on ice
throughout the experiment), the assay was started by addition of
55Fe-siderophore (1 mL at 0.9 µM). Aliquots (1 mL) were removed
at appropriate times, filtered through membrane filters (HAWP
Millipore, 0.45 µm pore size), and washed with 10 mL of cold 0.1
M sodium citrate. Filters were dried and 6 mL of liquid scintillation
Ecolume (ICN) was added. The vials were shaken, stored for 12 h,
and the radioactivity determined using a Perkin-Elmer Tri-Carb
2800 TR liquid scintillation analyzer.
Acknowledgment. We thank Dr. Youngchang Kim for many
helpful discussions, as well as Prof. John Helmann and Dr. Natalia
Maltseva for kindly providing us with bacterial strains and purified
Fes samples, respectively. This research was supported by the
National Institutes of Health (Grant AI11744).
Supporting Information Available: Additional figures and
spectral data obtained for both enzymatic and acidic hydrolysis
experiments; Synthetic scheme for D-SGC. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA903051Q
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12692 J. AM. CHEM. SOC. VOL. 131, NO. 35, 2009