Amino acid variability in the peptide composition of a suite of amphiphilic peptide siderophores from an open ocean Vibrio species

Article abstract of DOI:10.1007/s00775-013-0995-3

In response to iron-depleted aerobic conditions, bacteria often secrete low molecular weight, high-affinity iron(III)-complexing ligands, siderophores, to solubilize and sequester iron(III). Many marine siderophores are amphiphilic and are produced in suites, wherein each member within a particular suite has the same iron(III)-binding polar head group which is appended by one or two fatty acids of differing length, degree of unsaturation, and degree of hydroxylation, establishing the suite composition. We report the isolation and structural characterization of a suite of siderophores from marine bacterial isolate Vibrio sp. Nt1. On the basis of structural analysis, this suite of siderophores, the moanachelins, is amphiphilic and composed of two N-acetyl-N-hydroxy-d- ornithines, one N-acetyl-N-hydroxy-l-ornithine, and either a glycine or an l-alanine, appended with various saturated and unsaturated fatty acid tails. The variation in the small side-chain amino acid is the first occurrence of variation in the peptidic head group structure of a set of siderophores produced by a single bacterium.

Full text of DOI:10.1007/s00775-013-0995-3

J Biol Inorg Chem (2013) 18:489–497
DOI 10.1007/s00775-013-0995-3
ORIGINAL PAPER
Amino acid variability in the peptide composition of a suite
of amphiphilic peptide siderophores from an open ocean
Vibrio species
•
Julia M. Gauglitz Alison Butler
Received: 12 February 2013 / Accepted: 11 March 2013 / Published online: 6 April 2013
Ó SBIC 2013
Abstract In response to iron-depleted aerobic conditions,
bacteria often secrete low molecular weight, high-affinity
iron(III)-complexing ligands, siderophores, to solubilize
and sequester iron(III). Many marine siderophores are
amphiphilic and are produced in suites, wherein each
member within a particular suite has the same iron(III)-
binding polar head group which is appended by one or two
fatty acids of differing length, degree of unsaturation, and
degree of hydroxylation, establishing the suite composi-
tion. We report the isolation and structural characterization
of a suite of siderophores from marine bacterial isolate
Vibrio sp. Nt1. On the basis of structural analysis, this suite
of siderophores, the moanachelins, is amphiphilic and
composed of two N-acetyl-N-hydroxy-D-ornithines, one N-
acetyl-N-hydroxy-L-ornithine, and either a glycine or an L-
alanine, appended with various saturated and unsaturated
fatty acid tails. The variation in the small side-chain amino
acid is the first occurrence of variation in the peptidic head
group structure of a set of siderophores produced by a
single bacterium.
Keywords Marine siderophores Á Amphiphilic
siderophores Á Peptide-based Á Amphibactins Á
Moanachelins
Introduction
Siderophores are an important group of high-affinity iron-
coordinating molecules which are synthesized by bacteria
under iron-limited conditions to facilitate the uptake of
iron(III). Siderophores are composed of a variety of dif-
ferent building blocks, including amino acids, diamines,
citric acid, succinic acid, and dihydroxybenzoic acid. Sid-
erophores coordinate iron(III) through hydroxamic acid,
catechol, or a-hydroxy carboxylic acid ligation, with
exceptional affinity. A bacterium can produce a single
siderophore, such as desferrioxamine G produced by Vibrio
sp. BLI-41 [1], multiple siderophores such as petrobactin
and bacillibactin, both produced by various Bacillus spe-
cies [2], or an entire suite of structurally related amphi-
philic siderophores, where the difference lies in the nature
of the fatty acid appendage [3–9].
Julia M. Gauglitz previously published under the name Julia M.
Vraspir.
Any opinions, findings, conclusions, or recommendations expressed
in this publication are those of the authors and do not necessarily
reflect the views of the National Science Foundation.
A particularly interesting class of siderophores is com-
posed of suites of amphiphilic peptide-based siderophores.
This class of siderophores is produced by bacteria found in
both terrestrial (e.g., mycobactins, serobactins) and marine
(e.g., aquachelins, marinobactins, amphibactins) environ-
ments (Fig. 1). Amphiphilic peptide-based siderophores
have been isolated from pure bacterial cultures, and have
been identified as products from nutrient-enriched seawater
incubations [10, 11]. Specifically, siderophore-like chelates
with the same exact mass as well as the appropriate mass
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-0995-3) contains supplementary
material, which is available to authorized users.
J. M. Gauglitz
Graduate Program in Marine Science, University of California,
Santa Barbara, CA 93106, USA
J. M. Gauglitz Á A. Butler (&)
Department of Chemistry and Biochemistry, University of
California, Santa Barbara, CA 93106, USA
e-mail: butler@chem.ucsb.edu
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J Biol Inorg Chem (2013) 18:489–497
Marinobactins
Aquachelins
Amphibactins
O
O
O
O
O
O
OH
N
N
N
N
N
N
H
O
HO
HO
HO
HO
HO
HO
N
HO
O
HO
OH
O
O
O
O
O
O
O
R
H
H
H
H
H
H
H
R
N
N
OH
N
N
N
OH
N
N
N
O
O
N
H
N
N
H
N
H
OH
N
N
N
N
N
H
R
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
OH
O
H2N
O
=
R
N
O
OH
=
R
O
O
O
O
O
O
R:
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
O
O
OH
Serobactins
Mycobactins
OH
R4
OH
O
O
R2
O
HO
HO
O
H
N
O
O
OH
O
O
N
OH
O
O
N
O
N
H
H
H
R
H
N
N
N
OH
OH
O
N
H
N
N
N
R3
O
O
H
H
O
O
O
OH
HO
N
R1
O
HO
=
R
O
O
12CH
Mycobactin J: R1 = -CH=CH(CH
2
)
3
R2 = -CH(CH3)
R3 = -CH3
R4 = -H
2
Mycobactin
2 8-16CH3
)
F(a-e): R1 = -(CH
= -CH3
R2
)
Mycobactin T: R1 = -(CH
2
18CH3
R3 = -H
R2 = -CH3
R3 = -H
R4 = -CH3
R4 = -H
Fig. 1 Select peptide-based siderophore suites. Terrestrial: mycobactins [8, 12, 13] and serobactins [14]. Marine: aquachelins [7, 9],
marinobactins [7], and amphibactins [6, 9]
fragmentation pattern of various amphibactin siderophores
were identified [11]. Although several of the amphibactins
identified in seawater incubation experiments (m/z 832 and
858) were previously characterized [6], the two uncharac-
terized amphibactins in the previous work (m/z 804 and
832) [11] have now also been isolated and characterized
[9].
acid, with downstream acceptance, can lead to the pro-
duction of a set of closely related molecules [21–23].
The NRPS-dependent biosynthetic pathways of a num-
ber of siderophores have been well studied, including those
of enterobactin [24–26], pyochelin [27], yersiniabactin
[28], vibriobactin [29], mycobactins [15], and pyoverdine
[30, 31]. The amphiphilic peptidic marine siderophores are
likely also assembled by NRPS multienzyme systems
owing to the inclusion of unique features such as D-amino
acids, N-terminally attached fatty acid chains, and hetero-
cyclic elements [23, 32]. Recent investigations of marine
metagenomes indicate marine prokaryotes may use NRPSs
in the biosynthesis of some siderophores [33].
The biosynthetic pathways of peptidic marine sidero-
phores have not been investigated; however, the myco-
bactins, which are also produced as a suite of siderophores
with differing fatty acid tails, are produced by a nonrib-
osomal peptide synthetase (NRPS)-dependent biosynthetic
pathway [8, 15, 16]. The NRPS adenylation domain (A-
domain) plays an important role in amino acid substrate
recognition and determining the order and identity of the
amino acids incorporated into the peptide product. The
selectivity of the A-domain is conferred by an approxi-
mately ten amino acid sequence binding site [17–20]. In
general, strict amino acid substrate specificity is observed
for NRPS A-domains. However, promiscuity in this
domain in terms of the identity of the adenylated amino
We report herein the isolation and structural charac-
terization of the moanachelins, a new suite of amphi-
philic siderophores produced by Vibrio sp. Nt1 isolated
from oligotrophic open ocean water. The moanachelins
are the first suite of siderophores which exhibit variation
not only in their fatty acid appendages, but also in the
amino acid composition of the peptidic head group
structure.
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J Biol Inorg Chem (2013) 18:489–497
Materials and methods
Bacterial strain
491
1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (Marfey’s
reagent) after hydrolysis of the siderophores with 55 %
hydroiodic acid (Spectrum Labs) at 110 °C for 24 h [34].
The hydrolyzed and derivatized amino acids were identi-
fied by co-injection with derivatized authentic amino acid
standards and by comparison of the retention times when
resolved by reversed-phase HPLC. The solvents used for
HPLC separation were 99.99 % ddH2O (Barnstead Nano-
pure II) with 0.01 % TFA and 100 % HPLC grade aceto-
nitrile (Fisher).
Vibrio sp. Nt1 was isolated from an oligotrophic surface
seawater sample collected on July 15, 2009 at 20.30020°N,
153.42077°W while we were aboard the R/V Kilo Moana
northeast of Hawaii (GenBank ID JQ996144). Bacterial
isolation was performed as previously described [9]. The
bacterium was identified by sequencing of the 16S small
subunit ribosomal RNA gene as described in Gauglitz et al.
[3]. Vibrio sp. HC0601C5 was also grown for isolation of
amphibactin D (Fig. 1, C14:0) for structural comparison
using previously published methods [9].
The fatty acids were identified by gas chromatography–
mass spectrometry (GC–MS; Varian Saturn 2100T). Sid-
erophores were incubated with 3 N methanolic hydro-
chloride (Sigma) at 110 °C for 3 h to hydrolyze the amide
bonds and generate the methyl esters of the fatty acid
appendages. The methyl esters analyzed by GC–MS were
identified by comparison with authentic fatty acid methyl
ester standards (methyl dodecanoate, methyl tetradecano-
ate, and methyl cis-9-hexadecenoate; Supelco) and by co-
injection.
Siderophore isolation
For siderophore production, Vibrio sp. Nt1 was cultured in
2 L natural seawater medium in 4-L acid-washed Erlen-
meyer flasks. Natural seawater medium consists of 1 g
ammonium chloride, 2 g Bacto casamino acids, and 0.1 g
glycerol phosphate per liter of aged natural seawater.
Cultures were grown on a rotary shaker (180 rpm) at room
temperature until late exponential phase (about 14–18 h)
before harvesting. Siderophores were isolated from the
culture supernatant using the methods described in Gau-
glitz et al. [3]. The siderophores were purified by reversed-
phase high-performance liquid chromatography (HPLC) on
a preparative C4 column (250 mm length 9 20 mm
diameter, Higgins) with either a gradient from 100 %
solvent A [0.05 % trifluoroacetic acid (TFA) in doubly
deionized water (ddH2O; Barnstead Nanopure II)] to
100 % solvent B [0.05 % TFA in 80 % HPLC grade
methanol (Fisher), 20 % ddH2O] over 37 min, holding at
100 % solvent B for an additional 10 min, or a gradient
from 50 % solvent A and 50 % solvent B to 100 % solvent
B over 15 min, holding at 100 % solvent B for an addi-
tional 10 min, using a dual-pump Waters HPLC system.
The eluent was continuously monitored at 215 nm and
compounds corresponding to the peaks were collected by
hand and stored on dry ice. If necessary, fractions were
ultrapurified on the same preparative C4 column using the
same program as before. Purified siderophores were
lyophilized and stored at -20 °C.
UV–vis spectroscopy of the ferric moanachelin gly-D
and ferric amphibactin D complexes was performed with a
Cary 300 Bio UV–vis spectrophotometer (Varian). 1H–13C
heteronuclear single quantum correlation spectroscopy,
correlation spectroscopy, and heteronuclear multiple bond
correlation nuclear magnetic resonance (NMR) spectra of
moanachelin gly-D and amphibactin D in dimethyl-d6
sulfoxide (DMSO-d6, 99.9 % Cambridge Isotopes) were
recorded with a Bruker 800-MHz NMR spectrometer. In
addition, total correlation spectroscopy was also performed
for amphibactin D.
Results
A new suite of amphiphilic siderophores, the moanache-
lins, was isolated from the open ocean bacterium Vibrio sp.
Nt1. The moanachelins are a group of closely related
structures with variation not only in the fatty acid
appendage of the siderophore, but also in the amino acid
composition of the iron-coordinating peptidic head group.
To differentiate between the two head groups, we refer to
the siderophores which contain glycine as the moanache-
lins gly-B through gly-E, and to those which contain ala-
nine as the moanachelins ala-B and ala-D. The different
members of the moanachelin suite of siderophores are
designated as moanachelin gly or moanachelin ala on the
basis of the unique amino acid present, and are further
differentiated by the length of the fatty acid tail. Mo-
anachelins gly-D and ala-D both have a C14:0 fatty acid, as
does amphibactin D. During separation and isolation from
the culture supernatant, the moanachelins behave similarly
to the amphibactins, which have been isolated recently
Structure determination
The masses of the siderophores and the siderophore frag-
ments were determined by electrospray ionization mass
spectrometry and tandem mass spectrometry with a Mi-
cromass Q-TOF II instrument (Waters).
The amino acid composition of the isolated siderophores
was determined using the chiral derivatizing agent
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from the supernatant of Vibrio sp. HC0601C5 bacterial
cultures [9] as well as other Vibrio sp. [6], and which
contain serine in place of alanine or glycine (present in the
moanachelins). The moanachelins are eluted as a cluster of
peaks around 80 % methanol, indicating their relative
hydrophobicity (Fig. 2).
that the suite of siderophores can be broken up into two
groups, depending on the identity of the second amino acid
from the C terminus, and that they form a suite of sidero-
phores with variations in the fatty acid tails (Fig. 5). Tan-
dem mass spectrometry revealed most the ‘‘y’’ (‘‘y’’ is
[y ? 2H]?) and b fragments expected for the moanache-
lins [36]. The ‘‘y’’ fragments 191, 248, and 420 m/z are
consistently observed for the moanachelins containing
glycine, whereas the ‘‘y’’ fragments 191, 262, and 434 m/
z are observed for the moanachelins containing alanine
(Table 1, Figs. 3, 4, S1–S4). The identical ‘‘y’’ fragments
but different b fragments for each moanachelin (for both
those containing a glycine or an alanine residue) indicate
that the suite contains members with distinct fatty acid
appendages. For instance, the ‘‘y’’ fragments 191, 248, and
420 m/z, depicted in Fig. 6, are observed in the tandem
mass spectrum of moanachelin gly-D (Fig. 3). In addition
to moanachelins gly-B, gly-C, gly-D, gly-E, ala-B, and ala-
D, additional small quantities of other members of the suite
were also detected on the basis of their fragmentation
patterns, including 732 m/z (initially named moanachelin
gly-A) and 786 and 842 m/z. However, sufficient quantities
were not obtained to verify their structures.
A minimum of six moanachelins are produced. The
[M ? H]? molecular ions of the moanachelins fall within
the range of 774–828 m/z. Starting with the most hydro-
philic siderophore, moanachelin gly-B, the dominant sid-
erophore identified in peak 1 (Fig. 2), has an m/z ratio of
774.4. The dominant siderophores identified in peaks 2 and
3 are moanachelin ala-B (788.4 m/z) and moanachelin gly-
C (800.4 m/z), respectively. The most abundant sidero-
phore and the dominant siderophore in peak 4 is mo-
anachelin gly-D (802.4 m/z). The later part of peak 4 has
two small shoulder peaks, peaks 5 and 6, containing mo-
anachelin ala-D and moanachelin gly-E (816.5 and
828.5 m/z, respectively). All of these compounds had
positive responses to the liquid chrome azurol S test,
indicating their ability to coordinate iron(III) [35].
The fragmentation patterns seen in the tandem mass
spectra (Figs. 3, 4) and summarized in Table 1 indicate
Fig. 2 Reversed-phase high-
performance liquid
chromatography chromatogram
for total XAD extract of a
Vibrio sp. Nt1 culture using a
preparative Higgins C4 column.
The sample was monitored at
215 nm. Siderophores were
eluted between 47 and 53 min.
Peak numbers 1–6 are noted
Fig. 3 Electrospray ionization
tandem mass spectrum of
moanachelin gly-D (C14:0)
Vraspir, NT1 NSW peak 4; MSMS 802.5
BUT111610MC5 58 (1.103) Cm (1:60)
TOF MSMS 802.50ES+
1.28e3
420.22
100
%
341.29
802.52
421.23
248.13
383.31
803.52
323.28
402.21
342.30
425.31
230.13
191.11
612.40
249.14
555.38
131.08
784.51
0
m/z
150
200
250
300
350
400
450
500
550
600
650
700
750
800
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J Biol Inorg Chem (2013) 18:489–497
493
Fig. 4 Electrospray ionization
tandem mass spectrum of
moanachelin ala-D (C14:0)
Vraspir, Nt1 NSW, peak 5; MSMS 816.4
BUT120110MN5 88 (1.667) Cm (43:88)
TOF MSMS 816.40ES+
2.48e3
434.20
100
626.39
%
816.50
383.27
555.36
627.39
262.13
435.21
341.27
323.25
556.36
384.28
244.12
628.40
798.49
436.21
263.13
191.09
513.34
0
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
Table 1 The b and ‘‘y’’ fragment values (m/z) observed by tandem
mass spectrometry
bond was not determined, the identification was confirmed
by co-injection with fatty acid methyl ester standards
(Supelco). The NMR analysis of moanachelin gly-D is
consistent with this fatty acid tail analysis (Table 2).
Isolation procedures and structural analysis of the
moanachelins indicate that they are structurally similar to
the amphibactins, differing in the second amino acid
from the C terminus. Like the moanachelins, the am-
phibactins are produced as a suite of siderophores with a
conserved head group composed of two N-acetyl- N-
hydroxy-D-ornithines, one N–acetyl-N–hydroxy-L-orni-
thine, one L-serine, and a fatty acid appendage ranging
from C12 to C18 [6, 9]. NMR analyses of the amphi-
bactins and moanachelins in DMSO-d6 (D, 99.9 %) are
presented here to confirm the structure and connectivity
of these molecules.
Moanachelin
Gly-B
(774 m/z) (788 m/z) (800 m/z) (802 m/z) (816 m/z) (828 m/z)
Ala-B
Gly-C
Gly-D
Ala-D
Gly-E
C12:0
C12:0
C14:1
C14:0
C14:0
C16:1
b
b1 355
355
527
598
381
553
610
383
555
612
383
555
626
409
581
638
b2 527
b3 584
‘‘y’’
y1 191
191
262
434
191
248
420
191
248
420
191
262
434
191
248
420
y2 248
y3 420
‘‘y’’ is [y ? 2H]?
A side-by-side comparison of the head group chemical
1
shift values from the H and 13C NMR spectra of mo-
Chiral amino acid analysis of members of the mo-
anachelin suite confirmed the presence of two unique
combinations of amino acids. The amino acids were
identified by co-injection with authentic amino acid stan-
dards and by comparison of the retention times of the
amino acid standards. Moanachelins gly-B, gly-C, gly-D,
and gly-E are composed of two modified D-ornithines, one
modified L-ornithine, and one glycine (Fig. 5). Moanach-
elins ala-B and ala-D are composed of two modified
D-ornithines, one modified L-ornithine, and one L-alanine
(Fig. 5). The specific placement of the D-ornithines and
L-ornithines was not determined.
anachelin gly-D and amphibactin D is presented in Table 2.
Both siderophores are composed of a four amino acid head
group with a tetradecanoic acid tail. Tandem mass spectral
data as well as determination of the amino acid composi-
tion indicate that the structures of the two siderophores
differ only in the second amino acid from the C terminus.
Where the amphibactins have a serine residue [6], the
moanachelins have either a glycine residue or an alanine
residue. Comprehensive 1D and 2D NMR spectra allowed
the first full structural characterization of the moanachelins
(moanachelin gly-D; C14:0) and the amphibactins (am-
phibactin D; C14:0) (Figs. S7–S11 and S12–S17, respec-
tively). The numbering for the chemical shift assignments
for moanachelin gly-D is given in Fig. 7. The chemical
The suite of moanachelin siderophores was hydrolyzed
and derivatized with methanolic hydrochloride, extracted
into hexanes, and analyzed by GC–MS to determine the
identity of the fatty acids (Fig. 5). With the exception of
moanachelin gly-C, for which the position of the double
1
shifts of DMSO-d6 are noted at dH 2.49 in the H NMR
spectrum and dC 39.5 in the 13C NMR spectrum, and were
used as internal standards.
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Fig. 5 Suite of moanachelins.
The position of the double bond
in moanachelin gly-C was not
determined
O
O
N
N
HO
HO
O
O
H
HN
N
OH
R2
N
N
H
H
O
R1
O
OH
N
O
R2 =
gly-E (828 m/z; R1=H; R2 = C16:19)
ala-D (816 m/z; R1=CH3; R2 = C14:0)
gly-D (802 m/z; R1=H; R2 = C14:0)
gly-C (800 m/z; R1=H; R2 = C14:1)
ala-B (788 m/z; R1=CH3; R2 = C12:0)
gly-B (774 m/z; R1=H; R2 = C12:0)
O
O
O
O
O
?
O
420
O
248
= [y+2H]+
191
1
1
The H–1H correlation spectroscopy and H–13C heter-
onuclear multiple bond correlation spectra reveal an alkyl
group starting with the triplet methyl signal, H37, through
to the methylene resonance H25. The number of carbon
correlations to the alkyl chain seen in the H–13C hetero-
nuclear single quantum correlation spectroscopy and 13C
NMR spectra confirm the finding of the fatty acid analysis
that the alkyl group is a tetradecanoic acid. Through
examination of the 1D and 2D NMR data, the connectivity
of the molecule has been established (Table S1). Similar
analysis for amphibactin D reveals a remarkably related
structure (Table S2).
O
N
N
HO
HO
1
O
O
H
H
N
N
OH
O
N
N
H
H
O
O
OH
O
N
b = 383
555
612
Fig. 6 ‘‘y’’ and b fragment analysis of moanachelin gly-D (C14:0).
‘‘y’’ is [y ? 2H]?
The key difference between the amphibactin and mo-
anachelin spectra lies with the Ca chemical shift of the
serine present in amphibactin D versus the glycine present
in moanachelin gly-D. The Ca carbon chemical shift for
serine is dC 55.4, whereas it is dC 41.37 for glycine. The Ca
for serine has only one proton, whereas glycine has two. In
addition to a difference in the chemical shift of the serine
Ca proton and carbon (C9 for amphibactin D and mo-
anachelin gly-D), there is the additional signal of the serine
beta carbon and protons (present in amphibactin D, C10, at
dC 61.68 and dH 3.55, 3.59) (Table 2). All further spectral
values are very similar.
1
The H NMR spectrum of moanachelin gly-D reveals
the presence of four amide protons at dH 7.93, dH 7.98, dH
8.1, and dH 8.11, three N–acetyl-N–hydroxyornithine Ca
protons at dH 4.15, dH 4.19, and dH 4.23, and the glycine Ca
protons at dH 3.73, dH 3.75, dH 3.66, and dH 3.68. The
triplet at dH 0.84 corresponds to the methyl protons of the
terminal carbon, and the aliphatic protons are located
between dH 1 and dH 2. The 13C spectrum reveals the
presence of four unique carbonyl signals at dC 168.6, dC
171.6, dC 172.0, and dC 173.3, and one signal at dC 170.2
representing the carbonyl of the three modified ornithine
side chains (C6, C15, C22). The carbon chemical shifts at
C6, C15, and C22 are likely identical because of the locally
identical surroundings of the N-acetylated, N-hydroxylated
ornithine moiety. The gamma carbons of the modified
ornithine residues are found at approximately dC 46.3.
These data, in combination with the tandem mass
spectral data and amino acid analysis, demonstrate that the
moanachelins are composed of two N–acetyl-N–hydroxy-
D-ornithines, one N–acetyl-N–hydoxy-L-ornithine, and
either a glycine or an L-alanine subunit, followed by a fatty
acid appendage ranging from C12 to C16 linked by an
amide bond. In addition, the UV–vis spectrum of iron(III)
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J Biol Inorg Chem (2013) 18:489–497
495
1
Table 2 Comparison of H and 13C chemical shifts of head group atoms for amphibactin D (C14:0) and moanachelin gly-D (C14:0)
Position
Amphibactin D head group
13C (ppm) 1H (ppm) [m]
Position
Moanachelin gly-D head group
13C (ppm) 1H (ppm) [m]
N–acetyl-N–hydroxyornithine
N–acetyl-N–hydroxyornithine
C1
173.2
51.41
28.38
22.9
46.5
170.2
20.07
–
–
C1
C2
C3
C4
C5
C6
C7
N1
Glycine
C8
C9
–
173.3
51.45
28.36
23.09
46.3
170.2
20.1
–
–
C2
4.13
4.15
C3
1.67
1.55
C4
1.54, 1.48 (d)
1.54, 1.47 (d)
C5
3.45
–
3.44
–
C6
C7
1.95
7.78
1.96
7.98
N1
Serine
C8
169.5
55.4
61.68
–
–
168.6
41.37
–
–
C9
4.26
3.75, 3.73; 3.68, 3.66 (dd)
C10
N2
3.55, 3.59 (d)
7.83
–
N2
–
8.1
N–acetyl-N–hydroxyornithine
N–acetyl-N–hydroxyornithine
C11
C12
C13
C14
C15
C16
C17
N3
171.43
52.5
28.9
22.9
46.5
170.2
20.07
–
–
C10
C11
C12
C13
C14
C15
C16
N3
171.6
52.16
29.0
22.89
46.31
170.2
20.1
–
–
4.2
4.19
1.46
1.46
1.54, 1.48 (d)
1.54, 1.47 (d)
3.47, 3.42 (d)
3.47, 3.49 (d)
–
–
1.95
8
1.96
8.11
N–acetyl-N-hydroxyornithine
N–acetyl-N–hydroxyornithine
C18
C19
C20
C21
C22
C23
C24
N4
172.89
52.1
28.8
22.9
46
–
C17
C18
C19
C20
C21
C22
C23
N4
172.0
52.28
29.12
23.0
46.31
170.2
20.1
–
–
4.23
4.23
1.68
1.46
1.54, 1.48 (d)
1.52, 1.47 (d)
3.44
–
3.47, 3.49 (d)
170.3
20.07
–
–
1.95
8.17
1.96
7.93
See the numbering in Fig. 7. The multiplicity is given in parentheses.
moanachelin gly D and that of iron(III) amphibactin D
reveal broad absorption maxima at 415 nm, characteristic
of the hydroxamate to iron(III) charge-transfer band (Figs.
S5, S6).
L-ornithine, and one variable amino acid, which form a
peptidic head group that binds iron(III). The moanachelins
are subcategorized depending on whether they contain
glycine or alanine in the moanachelins gly-B through gly-E
and moanachelins ala-B and ala-D. The existence of vari-
ation in the peptidic head group structure of a suite of
amphiphilic siderophores produced by a single bacterium is
unique to date.
Discussion
We have presented here the structures of the moanachelins,
a suite of amphiphilic marine siderophores produced by an
open ocean Vibrio species. This suite of siderophores has
differing fatty acid tail lengths and is composed of two N-
acetyl-N–hydroxy-D-ornithines, one N-acetyl-N–hydroxy-
Although the moanachelins have either glycine or
L-alanine at the same position in the peptide, no amino acid
variation has been observed in the L-serine-containing
amphibactins (see Figs. 1, 5). The exclusive presence of
L-serine in the amphibactins could be due to the highly
123

496
J Biol Inorg Chem (2013) 18:489–497
23
accommodated in downstream domains, allowing either
alanine or glycine to be incorporated into the siderophore.
Marine microbes have been shown to modify the
hydrophilicity of their amphiphilic siderophores by varying
the length, degree of unsaturation, and degree of hydrox-
ylation of their fatty acid appendages. The large suites of
amphiphilic siderophores that have been discovered in the
past decade indicate that there is broad selectivity for the
N-terminal fatty acid chain in the biosynthetic pathway,
and it remains to be seen if these suites confer a specific
advantage in an aqueous environment or if it is metaboli-
cally more favorable for the bacterium to incorporate a
range of fatty acid tails. These linear peptide siderophores
are produced by gammaproteobacteria isolated from the
marine environment and are produced by a number of
different genera. The continued detection of new sidero-
phores with common structural features, isolated from
numerous ocean environments, points toward an evolu-
tionary lineage to siderophore production.
O
22
N
21
OH
20
O
OH
1
19
18
H
N
H
N
3
1
R
10
9
17
11
N
H
8
2
3
O
N
2
4
H
O
12
O
13
14
4
5
HO
O
HO
O
N
N
6
15
16
7
R=
37
O
34
32
30
28
26
24
36
31
29
27
25
35
33
Fig. 7 1H and 13C number assignment for moanachelin gly-D. The
chemical structure of amphibactin D differs at the C9 alpha carbon,
where a serine group is present in contrast to the glycine depicted for
moanachelin gly-D. Therefore, the numbering is the same for C1 to
C8 and is shifted by 1 after C9 for amphibactin D because of the beta
carbon of serine (see Table 2)
Acknowledgments We thank the officers, crew, and scientific party
aboard the R/V Kilo Moana during the 2009 POOB cruise. A.B.
gratefully acknowledges support from the National Institutes of
Health (GM38130) and the National Science Foundation (CHE-
1059067). J.M.G. is supported by a National Science Foundation
Graduate Research Fellowship (2007052970).
conserved structure for the adenylation domain binding site
specificity pocket for L-serine, which would be present in
the NRPS system [19]. The glycine and alanine binding
sites, on the other hand, have not been as well character-
ized, and modeling studies indicate that they group into the
same ‘‘small cluster’’ [20]. This could indicate that they
have similar binding pockets and may substitute for one
another more readily or that there may be many degenerate
solutions for the glycine and alanine binding pockets and
multiple conformations of the binding pocket may recog-
nize these small amino acids.
References
1. Martinez JS, Haygood MG, Butler A (2001) Limnol Oceanogr
46:420–424
2. Wilson M, Abergel R, Arceneaux J, Raymond K, Byers B (2010)
Biometals 23:129–134
3. Gauglitz JM, Zhou H, Butler A (2012) J Inorg Biochem
107:90–95
4. Homann VV, Sandy M, Tincu J, Templeton A, Tebo BM, Butler
A (2009) J Nat Prod 72:884–888
5. Martin JD, Ito Y, Homann VV, Haygood MG, Butler A (2006) J
Biol Inorg Chem 11:633–641
The occurrence of bacterial peptidic secondary metab-
olites which naturally incorporate different but structurally
similar amino acids is rare. One instance is the production
of a pyoverdine siderophore with two structural variants,
whose amino acid structure differs in the incorporation of
alanine or glycine [30]. However, the substitution of gly-
cine was restricted to a specific alanine residue in the
siderophore and the glycine-containing siderophore was a
minor portion of the total siderophore production and could
not be independently isolated. In the case of the mo-
anachelins, the alanine- and glycine-containing sidero-
phores also had very similar physical properties. Flexibility
in the NRPS system may result in the production of the two
variants. Thus, it is plausible that either two separate bio-
synthetic pathways are present or that, in the case of an
NRPS system, promiscuity of the adenylation domain is
6. Martinez JS, Carter-Franklin JN, Mann EL, Martin JD, Haygood
MG, Butler A (2003) Proc Natl Acad Sci USA 100:3754–3759
7. Martinez JS, Zhang GP, Holt PD, Jung HT, Carrano CJ, Haygood
MG, Butler A (2000) Science 287:1245–1247
8. Snow G, White A (1969) Biochem. J 115:1031–1045
9. Vraspir JM, Holt PD, Butler A (2011) Biometals 24:85–92
10. Mawji E, Gledhill M, Milton JA, Zubkov MV, Thompson A,
Wolff GA, Achterberg EP (2011) Mar Chem 124:90–99
11. Gledhill M, McCormack P, Ussher S, Achterberg EP, Mantoura
RFC, Worsfold PJ (2004) Mar Chem 88:75–83
12. Ratledge C (2004) Tuberculosis 84:110–130
13. De Voss J, Rutter K, Schroeder B, Barry C (1999) J Bacteriol
181:4443–4451
14. Rosconi F, Davyt D, Martinez V, Martinez M, Abin-Carriquiry
JA, Zane H, Butler A, de Souza EM, Fabiano E (2013) Environ
Microbiol 15:916–927
15. Quadri L, Sello J, Keating T, Weinreb P, Walsh CT (1998) Chem
Biol 5:631–645
16. Vergne A, Walz A, Miller M (2000) Nat Prod Rep 17:99–116
123

J Biol Inorg Chem (2013) 18:489–497
497
17. Eppelmann K, Stachelhaus T, Marahiel M (2002) Biochemistry
41:9718–9726
27. Quadri LEN, Keating TA, Patel HM, Walsh CT (1999) Bio-
chemistry 38:14941–14954
18. Stachelhaus T, Mootz H, Marahiel M (1999) Chem Biol
6:493–505
28. Keating TA, Suo Z, Ehmann DE, Walsh CT (2000) Biochemistry
39:2297–2306
19. Challis GL, Ravel J, Townsend C (2000) Chem Biol 7:211–224
20. Rausch C, Weber T, Kohlbacher O, Wohlleben W, Huson D
(2005) Nucleic Acids Res 33:5799–5808
21. Qiao K, Zhou H, Xu W, Zhang W, Garg N, Tang Y (2011) Org
Lett 13:1758–1761
22. Crawford JM, Portmann C, Kontnik R, Walsh CT, Clardy J
(2011) Org Lett 13:5144–5147
23. Marahiel M, Essen L-O (2009) Methods Enzymol 458:337–351
24. Gehring AM, Mori I, Walsh CT (1998) Biochemistry
37:2648–2659
29. Keating TA, Marshall CG, Walsh CT (2000) Biochemistry
39:15522–15530
30. Barelmann I, Fernandez DU, Budzikiewicz H, Meyer J-M (2003)
Biometals 16:263–270
31. Schalk IJ, Guillon L (2012) Environ Microbiol. doi:10.1111/
1462-2920.12013
32. Sieber S, Marahiel M (2005) Chem Rev 105:715–738
33. Hopkinson BM, Barbeau KA (2012) Environ Microbiol
14:114–128
34. B’Hymer C, Montes-Bayon M, Caruso JA (2003) J Sep Sci
26:7–19
35. Schwyn B, Neilands J (1987) Anal Biochem 160:47–56
36. Roepstorff P, Fohlman J (1984) Biomed Mass Spectrom 11:601
25. Rusnak F, Faraci WS, Walsh CT (1989) Biochemistry
28:6827–6835
26. Shaw-Reid CA, Kelleher NL, Losey HC, Gehring AM, Berg C,
Walsh CT (1999) Chem Biol 6:385–400
123