Enzymatic tailoring of ornithine in the biosynthesis of the rhizobium cyclic trihydroxamate siderophore vicibactin

Article abstract of DOI:10.1021/ja9056008

To acquire iron, the N₂-fixing, symbiotic bacterium Rhizobium sp. produce the cyclic trihydroxamate siderophore vicibactin, containing a 30-membered trilactone scaffold. Herein we report the overproduction and purification of the six proteins V

Full text of DOI:10.1021/ja9056008

Published on Web 09/24/2009
Enzymatic Tailoring of Ornithine in the Biosynthesis of the
Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
John R. Heemstra, Jr., Christopher T. Walsh,* and Elizabeth S. Sattely
Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School,
Boston, Massachusetts 02115
Received July 7, 2009; E-mail: christopher_walsh@hms.harvard.edu
Abstract: To acquire iron, the N2-fixing, symbiotic bacterium Rhizobium sp. produce the cyclic trihydroxamate
siderophore vicibactin, containing a 30-membered trilactone scaffold. Herein we report the overproduction
and purification of the six proteins VbsACGOLS in the bacterial host Escherichia coli and the reconstitution
of the biosynthesis of vicibactin from primary metabolites. The flavoprotein VbsO acts as a pathway-initiating
L-ornithine N5-hydroxylase, followed by VbsA, which transfers (R)-3-hydroxybutyryl- from the CoA thioester
to N5-hydroxyornithine to yield N5-((R)-3-hydroxybutyryl)-N5-hydroxy-L-ornithine. VbsL is a PLP-dependent
epimerase acting at C2 of the 10 atom monomer unit. VbsS, a nonribosomal peptide synthetase free-
standing module, then activates N5-((R)-3-hydroxybutyryl)-N5-hydroxy-D-ornithine as the AMP anhydride
on the way to cyclotrimerization to the vicibactin scaffold. The last step, tris-acetylation of the C2 amino
group of desacetyl-D-vicibactin to the mature siderophore vicibactin, is catalyzed distributively by VbsC,
using three molecules of acetyl-CoA.
(NRPS) assembly lines.8 Hydroxamates also appear to originate
from primary metabolic precursors, for example ornithine and
lysine; however, the pathway for elaboration to the mature
hydroxamate-containing siderophore is not as obvious.
Introduction
Iron is required for growth of nearly all living organisms.1
To cope with the often limited availability of soluble iron in
microenvironments, bacteria and fungi2 maintain a branch of
secondary metabolism for the production of Fe(III)-scavenging
small molecule siderophores. In response to iron starvation, these
microbes turn on genes that encode for the biosynthesis3 and
export of apo-siderophores and also for the uptake and ac-
cumulation of the iron-loaded forms.1 Siderophores are com-
monly made up of peptides decorated with iron-chelating
functionality of four main classes: (1) phenols and catechols,
(2) heterocyclic oxazolines and thiazolines, (3) hydroxamates,
and (4) the R-hydroxyl-carboxylates.1,4
The dominant logic for the biosynthesis of phenolic and
catecholic siderophores such as enterobactin5 involves amine
acylation, while heterocycles such as the thiazolines of yers-
iniabactin6 and pyochelin7 are derived from modified cysteine
or serine residues. Each of these tailoring events is known to
occur through the action of nonribosomal peptide synthetase
Despite the structural similarities of fungal fusarinines,9,10
bacterial vicibactins,11,12 and desferrioxamines13 exemplified in
Figure 1, two parallel strategies appear to have evolved for the
biosynthesis of these related hydroxamate siderophores. While
each macrocycle consists of a trimerized monomer, it is thought
that different classes of ATP-dependent synthetases are generally
utilized for the synthesis of ester verses amide-containing
siderophores.14 For example, in desferrioxamine E production
by Streptomyces coelicolor, the enzyme DesD uses ATP to
activate N-hydroxy-N-succinylcadaverine units as the mixed
anhydride (with release of PPi) and catalyzes trimerization of
soluble intermediates through a stepwise mechanism15 to
generate the 33-membered macrocyclic triamide.16 In contrast,
the pathogenic mold Aspergillus fumigatus makes virulence-
related siderophores fusarinine C and the triacetylfusarinine C
derivative as 36-membered marocyclic trilactones (depsipep-
(8) Fischbach, M. A.; Walsh, C. T. Chem ReV 2006, 106, 3468–3496.
(9) Hissen, A. H.; Wan, A. N.; Warwas, M. L.; Pinto, L. J.; Moore, M. M.
Infect. Immun. 2005, 73, 5493–5503.
(1) Miethke, M.; Marahiel, M. A. Microbiol. Mol. Biol. ReV. 2007, 71,
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F. R.; Collison, D.; Taylor, R. J. Mycol. Res. 2002, 106, 1123–1142.
(3) Barry, S. M.; Challis, G. L. Curr. Opin. Chem. Biol. 2009, 13, 205–
215.
(10) Eisendle, M.; Oberegger, H.; Zadra, I.; Haas, H. Mol. Microbiol. 2003,
49, 359–375.
(11) Carter, R. A.; Worsley, P. S.; Sawers, G.; Challis, G. L.; Dilworth,
M. J.; Carson, K. C.; Lawrence, J. A.; Wexler, M.; Johnston, A. W.;
Yeoman, K. H. Mol. Microbiol. 2002, 44, 1153–1166.
(12) Eng-Wilmot, D. L.; Rahman, A.; Mendenhall, J. V.; Grayson, S. L.;
van der Helm, D. J. Am. Chem. Soc. 1984, 106, 1285–1290.
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Challis, G. L. J. Am. Chem. Soc. 2004, 126, 16282–16283.
(14) Challis, G. L. Chembiochem 2005, 6, 601–11.
(4) Crosa, J. H.; Walsh, C. T. Microbiol. Mol. Biol. ReV. 2002, 66, 223–
249.
(5) Gehring, A. M.; Mori, I.; Walsh, C. T. Biochemistry 1998, 37, 2648–
2659.
(6) Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chem.
Biol. 2002, 9, 333–44. Miller, D. A.; Luo, L.; Hillson, N.; Keating,
T. A.; Walsh, C. T. Chem. Biol. 2002, 9, 333–344.
(7) Patel, H. M.; Tao, J.; Walsh, C. T. Biochemistry 2003, 42, 10514–
10527. Patel, H. M.; Tao, J.; Walsh, C. T. Biochemistry 1998, 37,
2648–2659.
(15) Kadi, N.; Arbache, S.; Song, L.; Oves-Costales, D.; Challis, G. L.
J. Am. Chem. Soc. 2008, 130, 10458–10459.
(16) Kadi, N.; Oves-Costales, D.; Barona-Gomez, F.; Challis, G. L. Nat.
Chem. Biol. 2007, 3, 652–656.
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A R T I C L E S
Heemstra et al.
fusarinine is the trihydroxamate vicibactin,18,19 produced by the
N2-fixing, pea symbiotic bacterium Rhizobium leguminosarum.20
Although both siderophores are derived from similarly elabo-
rated monomers, vicibactin harbors an additional degree of
complexity in the D-isomer of Orn. In addition, the core
vicibactin NRPS, VbsS,11 presumably responsible for the
assembly of the 30-membered trilactone, has a distinct domain
organization that differs markedly from the fungal assembly line
SidD. Following trimerization of N5-((R)-3-hydroxybutyryl)-N5-
hydroxy-D-ornithine, presumably activated as the phosphopan-
tetheinyl thioester on VbsS, the macrocyclic scaffold is further
tailored through 3-fold N-acetylation at the N2-amino groups.
It is possible that this final modification helps to curtail unwanted
acyl transfer reactions or to block the association of positive
charge with the mature siderophore. Despite the suggested role
and known requirement of VbsS in vicibactin assembly,11 details
of the timing and mechanism of monomer provision are not
known.
The nitrogen-fixing ability of Rhizobia sp. has enabled use
of these microbes as “biofertilizers”,21 especially for leguminous
plants. In return for a source of usable nitrogen, plants provide
root nodules and nutrients for Rhizobia colonization.22 The
multiple FeS clusters essential to endogenous nitrogenase
catalysis23 presents Rhizobia with a particular need for iron
accumulation. It has been observed that vicibactin-mediated
collection of iron from the rhizosphere is an important deter-
minant of productive symbiosis.24
In 2002 Carter11 and co-workers identified a cluster of eight
genes for vicibactin biosynthesis, VbsGSO, VbsADL, VbsC and
VbsP, in Rhizobium leguminosarum, assigned functions by a
combination of bioinformatic predictions and genetic knockouts
of each of the VbsACGLOS genes, and proposed a pathway that
features the single NRPS module protein VbsS as the central
catalyst (Figure 2). Bioinformatic analysis of the fully sequenced
relative Rhizobium etli CFN4225 revealed the presence of an
analogous gene cluster. In this study we have expressed and
purified the six enzymes VbsA, C, L, O, and VbsGS from R.
etli CFN42 in E. coli and have established catalytic activities
that provide insights into the timing of ornithine N5-hydroxy-
lation and acylation, the construction of the ten atom N5-((R)-
3-hydroxybutyryl)-N5-hydroxy-D-ornithine monomer unit, its
activation by the adenylation (A) domain of the four domain
(C-A-T-TE) VbsS NRPS single module subunit, and the tris-
acetylation at N2 of desacetyl-D-vicibactin.
Figure 1. (A) Structures of bacterial (vicibactin and desferrioxamine E)
and fungal (fusarinines) trimeric hydroxamate siderophores. (B) X-ray
structure of the neurosporin-Fe complex. Neurosporin, isolated from the
fungus, Neurospora, is identical to vicibactin. (Neurosporin is a single
example of a fungal siderophore containing D-Orn residues)
Materials and Methods
tide).17 The fusarinine C scaffold is assembled by the action of
the multidomain nonribosomal peptide synthetase SidD that also
utilizes ATP-dependent substrate activation. Rather than linking
soluble monomers as in the case of DesD, the fusarinine
mechanism for trimerization is thought to occur through the
successive condensation of N5-cis-anhydromevalonyl-N5-hy-
droxy-L-ornithine covalently templated on the pantetheinyl arm
of the synthetase thiolation domain.
Several chemical steps are required to transform an amine-
containing metabolite to an iron-chelating hydroxamic acid. In
the context of global strategies for siderophore biosynthesis,
we have been interested in the timing of biological N-
oxygenation verses N-acylation and the fate of the resulting
hydroxamate, including cyclotrimerization to trilactone macro-
cyclic scaffolds. A close relative to the A. fumigatus siderophore
Standard recombinant DNA, molecular cloning, and microbio-
logical procedures were performed according to the methods
(18) Dilworth, M. J.; Carson, K. C.; Giles, R. G. F.; Byrne, L. T.; Glenn,
A. R. Microbiology 1998, 144, 781–791.
(19) Carson, K. C.; Holliday, S.; Glenn, A. R.; Dilworth, M. J. Arch.
Microbiol. 1992, 157, 264–271.
(20) Barton, L. L.; Johson, G. V.; Bishop, Y. M. In Iron Nutrition in Plants
and Rhizospheric Microorganisms; Barton, L. L., Abadia, J., Eds.;
Springer: Netherlands, 2006; pp 199-214.
(21) Bloemberg, G. V.; Lugtenberg, B. J. Curr. Opin. Plant Biol. 2001, 4,
343–350.
(22) Stacey, G. In The Biological N-Cycle; Bothe, H., Ferguson, S. J.,
Newton, W. E., Eds.; Elsevier: New York, 2007; pp 147-164.
(23) Rubio, L. M.; Ludden, P. W. Annu. ReV. Microbiol. 2008, 62, 93–
111.
(24) Haas, H.; Eisendle, M.; Turgeon, B. G. Annu. ReV. Phytopathol. 2008,
46, 149–187.
(25) Gonzalez, V.; Santamaria, R. I.; Bustos, P.; Hernandez-Gonzalez, I.;
Medrano-Soto, A.; Moreno-Hagelsieb, G.; Janga, S. C.; Ramirez,
M. A.; Jimenez-Jacinto, V.; Collado-Vides, J.; Davila, G. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 3834–3839.
(17) Schrettl, M.; Bignell, E.; Kragl, C.; Sabiha, Y.; Loss, O.; Eisendle,
M.; Wallner, A.; Arst, H. N., Jr.; Haynes, K.; Haas, H. PLoS Pathog.
2007, 3, 1195–1207.
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Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
A R T I C L E S
Figure 2. Vicibactin gene cluster and annotated protein functions. The multidomain NRPS, VbsS, is thought to be the central catalyst responsible for
trimerization of the functionalized ornithine monomer. The N-terminal condensation domain (marked C*) of VbsS lacks required conserved residues in the
active site and is thought to be inactive. (A ) adenylation domain, T ) thiolation domain, TE ) thioesterase domain.)
described by Sambrook et al.26 E. coli Top10 (Invitrogen) was used
for routine cloning and propagation of DNA vectors. E. coli BL21
(DE3) (Invitrogen) transformed with pET-derived vectors (Novagen)
was used for overexpression in Luria-Bertani (LB) medium
supplemented with kanamycin. Oligonucleotide primers were
synthesized by Integrated DNA Technologies (Coralville, IA).
Phusion DNA polymerase, restriction enzymes, and T4 DNA ligase
were purchased from New England Biolabs. Recombinant plasmid
DNA was purified with a Qiaprep kit from Qiagen. Gel extraction
of DNA fragments and restriction endonuclease cleanup was done
with a QIAquick PCR cleanup kit from Qiagen. DNA sequencing
was performed at the Molecular Biology Core Facilities of the Dana
Farber Cancer Institute (Boston, MA). Nickel-nitrilotriacetic acid-
agarose (Ni-NTA) superflow resin and SDS-page gels were
purchased from Biorad. Protein samples were concentrated using
10 K mwco Amicon Ultra filters from Millipore.
HPLC analysis was performed on a Beckman System Gold
(Beckman Coulter) instrument on a Supleco Discovery C18 column
(250 × 4.6 mm). MS analysis of samples was carried out on an
Agilent 6210 Time-of-Flight MS with a dual nebulizer ESI source.
1H NMR spectra were recorded on a Varian 600 MHz spectrometer.
Chemical shifts are reported in ppm from tetramethylsilane with
the solvent resonance resulting from incomplete deuteration as the
internal standard (CDCl3 δ 7.26, D2O δ 4.75). Data are reported as
follows: chemical shift, multiplicity (s ) singlet, d ) doublet, t )
triplet, q ) quartet, br ) broad, m ) multiplet), coupling constants
(Hz), and integration. A Cary 50 Bio scanning spectrometer was
employed to record absorption spectra. Details for synthetic
substrate preparations are included in the Supporting Information.
Cloning, Overexpression, and Purification of VbsO, VbsA,
VbsL, and VbsC. The VbsO, VbsA, VbsL, and VbsC genes of the
vicibactin cluster were individually obtained by PCR amplification
with Phusion DNA polymerase of genomic DNA isolated from
Rhizobium etli CFN42 (ATCC51251). The VbsO gene was PCR
amplified using the forward primer 5′-aatcaatcatatgaccgtcgcttttac-
ccgc-3′ (NdeI restriction site underlined) and reverse primer 5′-
gatcaagcttggacgagagcatgccgtc-3′ (HindIII restriction site under-
lined). The VbsA gene was PCR amplified using the forward primer
5′-aatcaatcatatggatttcgatgccgccaga-3′ (NdeI restriction site under-
lined) and reverse primer 5′-gatcaagctttcatgatgccggcacccagag-3′
(HindIII restriction site underlined). The VbsL gene was PCR
amplified using the forward primer 5′-aatcaatcatatgaaccagtccttcagc-
ccc-3′ (NdeI restriction site underlined) and reverse primer 5′-
gatcaagctttcaccgcttcgcggcgtgcag-3′ (HindIII restriction site under-
lined). The VbsC gene was PCR amplified using the forward primer
5′-aatcaatcatatgagacgaacagtcgagatt-3′ (NdeI restriction site under-
lined) and reverse primer 5′-gatcaagctttcaaagcgcggtcctgctcag-3′
(HindIII restriction site underlined). The purified PCR products were
digested with NdeI and HindIII, ligated into similarly digested
pET24b (VbsO) or pET28a (VbsA, VbsL, and VbsC) expression
vectors using T4 DNA ligase (New England Biolabs). The
recombinant plasmids were propagated in E. coli TOP10 cells and
the identity of the resulting pET-24b (C-His6) and pET-28a (N-
His6) constructs were confirmed by DNA sequencing. The expres-
sion constructs were used to transform E. coli BL21 (DE3) cells,
grown to saturation in LB medium supplemented with 50 µg/mL
kanamycin at 37 °C, and diluted 1:100 into LB medium containing
50 µg/mL kanamycin. The cultures were incubated at 37 °C with
shaking, induced with 100 µM IPTG at OD600 ) 0.5-0.6, and then
incubated at 15 °C for ∼18 h. The cells were harvested by
centrifugation (6000 rpm × 6 min), resuspended in 30 mL of lysis
buffer (25 mM Tris, pH 8.0, 500 mM NaCl) and then lysed with
two passes through an Emulsiflex-C5 cell disruptor (Avestin). The
lysate was clarified by ultracentrifugation (35 000 rpm × 35 min).
The supernatant was incubated with Ni-NTA resin (0.25 mL/L
culture, prewashed with lysis buffer) for 1 h at 4 °C, and the mixture
was centrifuged (1800 rpm × 15 min). The unbound fraction was
discarded, and the resin was then transferred to a column and the
protein was eluted with an imidazole gradient using steps of 25
mL of 0 and 5 mM imidazole, 10 mL of 25 and 200 mM imidazole,
and 5 mL of 500 mM imidazole in lysis buffer. SDS-PAGE analysis
(4-15% Tris-HCl gel, BioRad) was employed to ascertain the
presence and purity of protein in each fraction. Fractions containing
pure protein were concentrated to 2 mL and subjected to gel
filtration on a Superdex 200 column connected to an Amersham
automated FPLC system at 4 °C (20 mM HEPES, pH 7.5, 80 mM
NaCl, 10% glycerol was used as the eluent). Fractions containing
the desired protein were concentrated with a Millipore 10 kDa MW
cutoff filter, flash frozen in liquid N2 as pellets, and stored at -80
°C. The concentration of purified protein solutions was determined
by the method of Bradford,26 with BSA as a standard (VbsO and
VbsL) or spectrophotometrically using calculated extinction coef-
ficients at (VbsA ε280 ) 37 930 M-1 cm-1 and VbsC ε280 ) 18 479
M-1 cm-1).
Cloning, Overexpression, and Purification of VbsGS. The
fragment encoding VbsGS was subcloned from genomic DNA
isolated from Rhizobium etli CFN42 (ATCC51251) as a bicistronic
operon using the forward primer 5′-aatcaatcatatggacaatcttgagccccgc-
(26) Sambrook, J.; Russell, D. W. Condensed Protocols from Molecular
Cloning: A Laboratory Manual; Cold Spring Harbor Press: New York,
2006.
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A R T I C L E S
Heemstra et al.
3′ (NdeI restriction site underlined) and the reverse primer 5′-
gatcaagctttgcgttatccttctctgt-3′ (HindIII restriction site underlined).
The transcript was amplified and ligated into similarly digested pET-
24b vector as described above to append a C-terminal His6 tag to
VbsS (VbsG untagged). The recombinant plasmid was propagated
in E. coli Top10 cells and confirmed by sequencing. After
transformation of E. coli BL21 with the VbsGS-containing plasmid,
two 2 L cultures of the resulting expression strain were grown at
25 °C in LB media (supplemented with 40 µg/mL kanamycin) with
shaking at 180 rpm to an OD600 of 0.3, and then at 15 °C to an
OD600 of 0.6 at which time expression was induced by the addition
of IPTG to a final concentration of 0.1 mM, and growth was
continued at 15 °C for 15 h before the cells were harvested. A
suspension of collected cells in lysis buffer (25 mL; 20 mM Tris,
0.5 M NaCl, pH 8) was lysed with two passes through an
Emulsiflex-C5 cell disruptor (Avestin). The lysate was clarified by
ultracentrifugation (35 000 rpm for 35 min), combined with
(NH4)2SO4 to a saturation of 20% at 4 °C with gentle stirring, and
further incubated for 1 h. The precipitated protein was separated
by ultracentrifugation (35 000 rpm for 35 min) and resuspended in
10 mL of lysis buffer at 4 °C. Gentle stirring and agitation with a
spatula over 30 min was used to encourage dissolution. The
resulting cloudy solution was filtered through a 45 µm Acrodisc
syringe filter and subjected to gel filtration on a Superdex 200
column connected to an Amersham automated FPLC system at 4
°C (20 mM Tris, 50 mM NaCl, 10% v/v glycerol, pH ) 8 was
used as the eluent). Fractions containing VbsS, coeluting with VbsG
(as indicated by SDS PAGE analysis with Coomassie staining and
in gel tryptic digest/MS sequencing), were combined, concentrated
to 6 mL with a Millipore 50 kDa MW cutoff filter, flash frozen in
liquid N2 as pellets, and stored at -80 °C; concentration was
determined spectrophotometrically by absorbance at 280 nm.
Assays for VbsO N-Hydroxylase Activity. To a solution of
L-ornithine or D-ornithine (500 µM), FAD (50 µM), NADPH (2
mM) and pH 8.0 Tris buffer (50 mM) at 25 °C was added VbsO
(5 µM) to initiate the reaction (total reaction volume of 50 µL).
After 20 min the reactions were halted with MeCN (100 µL), chilled
at -20 °C for 10 min and centrifuged at 13 000 rpm for 5 min.
The supernatant was withdrawn, combined with 25 µL of 200 mM
borate (pH 8.0), followed by 20 µL of 10 mM 9-fluorenylmethyl
chloroformate (Fmoc-Cl), and the reaction was allowed to proceed
at room temperature for 5 min. To remove any remaining
derivatizing reagent, 20 µL of 0.1 M 1-aminoadamantane was added
and the mixture was kept at room temperature for an additional 10
min. Conversion was determined by reverse phase HPLC analysis
on a Supleco Discovery C18 column (250 × 4.6 mm) using a
solvent gradient of 20 to 100% B over 40 min (solvent A, 0.1%
TFA/H2O; solvent B, 0.1% TFA/MeCN). Product identity was
confirmed by coelution with a N5-hydroxy-L-ornithine (L-3) syn-
thetic standard Fmoc-derivatized in the same manner and by ESI-
HRMS. No product formation was observed in control incubations
without NADPH or with enzyme inactivated by boiling.
of 50 uL). After 8 min, the reactions were stopped with 10% TFA
(50 µL), chilled at -20 °C for 10 min, and centrifuged at 13 000
rpm for 5 min. Conversion was determined by reverse phase HPLC
analysis on a Supleco Discovery C18 column (250 × 4.6 mm) using
a solvent gradient of 2 to 66% B over 20 min (solvent A, 0.1%
TFA/H2O; solvent B, 0.1% TFA/MeCN). The elution profile was
monitored at 220 nm. Product identity was confirmed by coelution
with N5-hydroxy-N5-((R)-3-hydroxybutyryl)-L- or D-ornithine 9
synthetic standards and by ESI-HRMS. No product formation was
observed in control incubations omitting VbsA or with enzyme
inactivated by boiling.
Kinetic Investigations of VbsA. VbsA acyltransferase activity
was measured at 25 °C in a 300 µL reaction volume containing
Tris-HCl (50 mM, pH 7.4), N5-hydroxy-D-ornithine (D-3, 0-3 mM)
or N5-hydroxy-L-ornithine (L-3, 0-1 mM), 5,5′-dithio-bis(2-nitro-
benzoic acid) (DTNB) (50 µM), and ((R)-3-hydroxybutyryl)-CoA
11 (300 µM). Assays were initiated by the addition of VbsA (17
nM). Activity was monitored continuously on a Cary 50 Bio
UV-visible spectrophotometer by following the increase in ab-
sorbance at 412 nm resulting from the reaction between the free
thiol of CoASH, generated by VbsA-catalyzed acyl transfer, and
DTNB. Initial velocities were calculated from the measured
absorbance change over a 0.5-2 min time frame using the
extinction coefficient 13 600 M-1 cm-1. Reactions were run in
triplicate and the initial velocity data were fitted to the Michaelis-
Menten equation in Kaleidagraph (Synergy Software) to obtain
estimates for kcat and Km.
Assay for VbsL Epimerase Activity. To a solution of N5-
hydroxy-N5-((R)-3-hydroxybutyryl)-D- or L-ornithine 9 (1 mΜ), PLP
(50 µM), and pH 7.75 HEPES buffer (50 mM) was added VbsL
(500 nΜ) to initiate the reaction (total reaction volume of 100 µL).
Reaction aliquots (25 µL) were quenched at t ) 0, 16, 32, and 64
min with MeCN (50 µL), chilled at -20 °C for 10 min and
centrifuged at 13 000 rpm for 5 min. The supernatant was
withdrawn and combined with 25 µL of 200 mM borate (pH 8.0).
Twenty-five microliters of 10 mM 9-fluorenylmethyl chloroformate
(Fmoc-Cl) was added and the reaction was allowed to proceed at
room temperature for 5 min. To remove any remaining derivatizing
reagent, 25 µL of 0.1 M 1-aminoadamantane was added and the
mixture was kept at room temperature for an additional 30 min.
Racemization was determined by reverse phase HPLC analysis on
a chiralcel OD-RH column using a solvent gradient of 80 to 100%
B over 6 min followed by 100% B for 10 min (solvent A, 0.1%
TFA/H2O; solvent B, 0.1% TFA/MeCN). The elution profile was
monitored at 263 nm. Product identity was confirmed by coelution
with a similarly Fmoc-derivatized N5-hydroxy-N5-((R)-3-hydroxy-
butyryl)-D- or L-ornithine standard 9. No racemization was observed
in control incubations omitting PLP or with enzyme inactivated
by boiling.
VbsL Deuterium Incorporation Experiments. A solution of
N5-hydroxy-N5-((R)-3-hydroxybutyryl)-L-ornithine 9 (4 mΜ) and
PLP (50 µM) in pH 7.75 HEPES buffer (50 mM) was prepared
(total reaction volume of 1 mL), flash frozen in liquid N2, and
lyophilized for 36 h. The resulting residue was taken up in 950 µL
D2O and incubated with VbsL (2 µM) at room temperature for 18 h.
The reaction was halted with 2 mL of MeCN, stored at -20 °C for
20 min and centrifuged at 13 000 rpm for 5 min. The supernatant
was withdrawn and combined with 1 mL of 200 mM borate (pH
8.0). Two hundred microliters of 10 mM 9-fluorenylmethyl chlo-
roformate (Fmoc-Cl) was added and the reaction was allowed to
proceed at room temperature for 5 min. To remove any remaining
derivatizing reagent, 200 µL of 0.1 M 1-aminoadamantane was
added and the mixture was kept at room temperature for an
additional 30 min. Racemization was determined by reverse phase
HPLC analysis on a chiralcel OD-RH column using a solvent
gradient of 80 to 100% B over 6 min followed by 100% B for 10
min (solvent A, 0.1% TFA/H2O; solvent B, 0.1% TFA/MeCN).
The elution profile was monitored at 263 nm. Product peaks were
Kinetic Investigations of VbsO. VbsO N-hydroxylase activity
was measured at 25 °C in a 300 µL reaction volume containing
Tris-HCl (50 mM, pH 8.0), L-ornithine (0.065-5 mM), FAD (50
µM), and NADPH (300 µM). Assays were initiated by the addition
of VbsO (250 nM). Activity was monitored continuously on a Cary
50 Bio UV-visible spectrophotometer by following the decrease
in absorbance at 340 nm resulting from the conversion of NADPH
to NADP+. Initial velocities were calculated from the measured
absorbance change over a 0.5-2 min time frame using the
extinction coefficient 6300 M-1 cm-1. Reactions were run in
triplicate and the initial velocity data were fitted to the Michaelis-
Menten equation in Kaliedagraph (Synergy Software) to obtain
estimates for kcat and Km.
Assays for VbsA Acyltransferase Activity. To a solution of
N5-hydroxy-L- or D-ornithine 3 (1.2 mΜ), ((R)-3-hydroxybutyryl)-
CoA 11 (1 mΜ), and pH 7.5 Tris buffer (50 mM) at 25 °C was
added VbsA (1 µM) to initiate the reaction (total reaction volume
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Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
A R T I C L E S
collected, flash frozen in liquid N2, lyophilized overnight, resus-
pended in MeOH, and analyzed by ESI-HRMS analysis.
Assays for VbsC Acetyltransferase Activity. To a solution of
desacetyl-D-vicibactin (D-14) (1 mΜ), acetyl-CoA (4 mΜ), and
pH 7.75 HEPES buffer (50 mM) was added VbsC (10 µM) to
initiate the reaction (total reaction volume of 350 uL). Reaction
aliquots (50 µL) were quenched at t ) 0, 1, 2, 4, 8, 16, and 32 min
with 10% TFA or 10 mM Fe(ClO)3 in 0.2 M perchloric acid (50
µL). Reactions halted with TFA were chilled at -20 °C for 10
min and centrifuged at 13 000 rpm for 5 min. Reactions halted with
Fe(ClO)3 were incubated at ambient temperature for 18 h and
centrifuged at 13 000 rpm for 5 min. Conversion was determined
by reverse phase HPLC analysis on a Supleco Discovery C18
column (250 × 4.6 mm) using a solvent gradient of 2 to 66% B
over 20 min (solvent A, 0.1% TFA/H2O; solvent B, 0.1% TFA/
MeCN). The elution profile was monitored at 220 nm for the
desferri-products and at 435 nm for the ferri-products. Product peaks
were collected, flash frozen in liquid N2, lyophilized overnight,
resuspended in MeOH, and analyzed by ESI-HRMS analysis.
ATP-PPi Exchange Assay to Determine Activity of the
VbsS A Domain. A typical experiment (700 µL total volume) was
carried as follows: MgCl2 (4 mM), TCEP (5 mM), ATP (5 mM),
tetrasodium[32P]pyrophosphate (1 mM, 1.2 µCi), and substrate
amino acid (5 mM) were combined in reaction buffer (75 mM Tris,
pH 7.5); reactions were then initiated by the addition of VbsGS to
a final concentration of 2.0 µM. At regular time intervals, 100 µL
aliquots (90 µL for T ) 0, before addition of enzyme) were
quenched with a 500 µL solution of activated charcoal (1.6% w/v),
200 mM tetrasodium pyrophosphate, and 3.5% perchloric acid in
water. The charcoal was pelleted by centrifugation and washed twice
with a 500 µL solution of 200 mM tetrasodium pyrophosphate and
3.5% perchloric acid in water. The radioactivity of ATP bound to
the charcoal was then measured by liquid scintillation counting.
Turnover was calculated as (% incorporation of 32P-PPi)[total PPi]/
[Enz].
Results
Isolation of Ferrivicibactin. Cell mass from a 50 mL culture
of R. etli CFN42 (OD600 2.0) grown in MSM minimal media
(prepared according to a procedure reported by Dilworth27) was
suspended in 50 mL fresh media and used to inoculate 500 mL
MSM minimal media. The resulting culture was grown at 30 °C
for 40 h to an OD600 of 1.8. The presence of vicibactin in the culture
supernatant during growth was monitored periodically by the Csa´ky
method as described in a report by Dilworth.19 The culture
supernatant was separated from cell debris and harvested by
centrifugation and subsequent filtration through a 45 µM filter.
According to the Csa´ky method,19 the concentration of vicibactin
in the filtrate was estimated to be 76 uM. To generate ferric
vicibactin for ease of isolation, 496 µL of Fe(NH4)2(SO4)2 ·6H2O
(0.1 M in H2O) was added and the resulting solution was stirred
for 12 h at ambient temperature at which time it took on an orange/
red color. The extraction of ferric vicibactin in benzyl alcohol was
then carried out as reported previously.28 The resulting crude
methanolic extract containing vicibactin was dissolved in 10 mL
H2O and purified in three portions by preparative HPLC (Phenom-
enex Luna C18, 250 × 21.2 mm, 10 µm, 100 A column, gradient
0 to 100% B (B ) MeOH with 0.1% TFA v/v, A ) H20 with
0.1% TFA v/v). The elution of ferric vicibactin at 19 min was
detected by UV absorbance at 435 nm. The combined vicibactin
containing fractions were concentrated and lyophilized to 8 mg of
a red powder.
Expression and Purification of VbsA, VbsC, VbsO, and
VbsL. The VbsA, VbsC, VbsO and VbsL genes were individually
amplified from genomic DNA isolated from R. etli CFN42 by
PCR and cloned into E. coli expression vectors as N- or
C-terminal His6 fusions. The proteins were overexpressed in E.
coli BL21(DE3) cells at 15 °C with 0.1 mM IPTG and purified
using Ni-NTA column chromatography and gel filtration in
tandem. VbsA (37.9 kDa, 329 aa) yielded 2.9 mg/L, VbsC (18.5
kDa, 164 aa) yielded 12.0 mg/L, VbsO (50.6 kDa, 453 aa)
yielded 0.6 mg/L, and VbsL (43.5 kDA, 396 aa) yielded 0.5
mg/L. All proteins were purified to near homogeneity with a
correct molecular weight as judged by SDS-PAGE (Figure S1,
Supporting Information). The flavin dependent monooxygenase
VbsO purified with FAD as bound cofactor, as determined by
analytical HPLC using authentic standards (data not shown).
However, using UV-vis spectroscopy, the FAD occupancy was
determined to be less than 5%.
Expression and Purification of VbsS and VbsG. The VbsGS
transcript was amplified from R. etli CFN42 genomic DNA and
cloned into a pET24b E. coli expression vector to append a
C-terminal His6 tag to VbsS (VbsG untagged). The proteins were
overexpressed in E. coli BL21(DE3) cells at 15 °C following
induction with IPTG, and purified by (NH4)2SO4 precipitation
and gel filtration in tandem. SDS-PAGE analysis of the eluted
fractions and in gel tryptic digest and MS sequencing (Figure
S2, Supporting Information) indicated that VbsG (8 kDa)
copurifies with the 147 kDa VbsS. Although the VbsGS
expression construct incorporates a C-terminal His6 tag, we
found that VbsS does not bind to Ni for affinity purification.
VbsS alone, either N or C-His tagged exhibits similar behavior;
however, in an initial screen, the VbsGS expression vector was
produced in the highest yield following (NH4)2SO4 precipitation;
therefore the two copurified proteins VbsGS were utilized for
activity assays.
Isolation of L- and D-Desacetylvicibactin. Culture supernatants
of J369 and J372,11 VbsC and VbsL mutants, respectively, were
prepared as reported above for WT R. etli CFN42 (growth in MSM
minimal media). Siderophore concentration was determined by the
Csa´ky method as for the detection of vicibactin. Two-hundred fifty
milliliters of filtered culture supernatant was concentrated in vacuo
to a white solid residue, from which desacetylvicibactin was
extracted with 40 mL of MeOH according to a modified literature
procedure.29 The resulting suspension was filtered through a sintered
glass frit and again concentrated in vacuo. The extraction procedure
was then repeated a second time. Finally, the resulting residue was
dissolved in 20 mL 1:1 H2O:MeOH and purified by preparative
HPLC in four portions (Phenomenex Luna C18, 250 × 21.2 mm,
10 µm, 100 A column, gradient 0 to 100% B (B ) MeOH with
0.1% TFA v/v, A ) H20 with 0.1% TFA v/v). Desferri-de-
sacetylvicibactin elutes at 17.3 min with UV detection (220 nm).
The combined desacetylvicibactin fractions were concentrated and
lyophilized to give 16 mg of L-desacetylvicibactin from J372.
Similar yields were obtained for D-desacetylvicibactin from J369.
Synthesis of N5-Hydroxy-D- and L-Ornithine (D- and L-3)
and N5-((R)-3-Hydroxybutyryl)-N5-Hydroxy-D- and L-Orni-
thine (D- and L-9). The requisite hydroxylamine substrates were
prepared using an indirect oxidation method developed by Miller
and co-workers30 (Scheme 1). Starting with commercially
available N2-Boc-D- or L-ornithine (1), the primary amine was
converted to an imine followed by the mCPBA oxidation to
the oxaziridine. Treatment with TFA isomerized the oxaziridine
to a nitrone and afforded amino acids D- and L-2 in 45% and
1
The H NMR spectrum of isolated material was analogous to that
reported previously.18
(27) Brown, C. M.; Dilworth, M. J. J. Gen. Microbiol. 1975, 86, 39–48.
(28) Carson, K. C.; Glenn, A. R.; Dilworth, M. J. Arch. Microbiol. 1994,
161, 333–339.
(29) Emery, T. Biochemistry 1965, 4, 1410–1417.
(30) Lin, Y.-M.; Miller, M. J. J. Org. Chem. 1999, 64, 7451–7458.
9
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A R T I C L E S
Heemstra et al.
Scheme 1. Synthesis of Hydroxylamines L- and D-3a
Characterization of VbsO. The product of the VbsO gene is
homologous to IucD,33 CchB34 and PvdA,35 flavin-dependent
monooxygenases36 from E. coli, S. coelicolor and P. aeruginosa
respectively. IucD catalyzes the regiospecific hydroxylation of
the ε-amino group of L-lysine as the first step in biosynthesis
of the hydroxamate containing siderophore aerobactin, while
CchB and PvdA carry out hydroxylation of the δ-amino group
of L-ornithine as the first committed step in coelichelin and
pyoverdine biosynthesis respectively. Therefore, we anticipated
that VbsO-mediated N5-hydroxylation of ornithine would be the
starting point in the biosynthesis of vicibactin as well.
a Yields shown in parentheses.
To evaluate the catalytic activity of VbsO, the recombinant
enzyme was incubated with L-Orn in the presence of FAD and
NADPH. To facilitate identification and detection of the
hydroxylated amino acid, the assays were derivatized with
9-fluorenylmethyl chloroformate (Fmoc-Cl) to yield highly UV
active Fmoc derivatives. As shown in figure 3, the enzymatic
incubation with L-Orn and subsequent derivatization with Fmoc-
Cl resulted in the formation of a new peak in the analytical
HPLC trace with a retention time consistent with authentic N5-
hydroxy-L-Orn bisFmoc standard, bisFmoc-L-3. Further con-
firmation of product formation was obtained by ESI-HRMS
analysis ([M+Na]+ m/z calcd., 615.2107; found, 615.2114) of
the new species purified by HPLC (Figure S3, Supporting
Information). No activity was detected with enzyme inactivated
by boiling or in the absence of NADPH (data not shown).
The Michaelis-Menten kinetic parameters of VbsO for L-Orn
hydroxylation were determined to an apparent kcat of 108 ( 2
min-1 and KM of 0.305 ( 0.024 mM, as determined by
monitoring the conversion of NADPH to NADP+ at 340 nm
(Figure 3C). For comparison, the apparent KM value of VbsO
for L-Orn is similar to the reported KM values of PvdA35 for
L-Orn (0.593 mM) and IucD33 for L-Lys (0.11 mM) and 10-
fold lower than the reported KM value of CchB34 for L-Orn (3.6
mM). Regarding the catalytic efficiencies (kcat/Km) of the three
L-ornithine N5-hydroxylases, VbsO shows a 10-fold higher
efficiency (354.10 min-1 mM-1) than PvdA (44.52 min-1 mM-1)
Scheme 2. Synthesis of Hydroxamates D- and L-9a
a Yields shown in parentheses.
42% respectively over three steps. Finally, acid catalyzed
hydrolysis of the nitrone afforded the HCl salts of D-3 in 35%
yield and L-3 in 78% yield.
and
a hundredfold higher efficiency that CchB (4.83
min-1 mM-1).
To probe the substrate specificity of the enzyme, we tested
D-Orn, L- or D-Lys, and N5-((R)-3-hydroxybutyryl)-L- or D-Orn
(L- or D-10) for VbsO-mediated hydroxylation by spectropho-
tometric measurement of NADPH oxidation and HPLC product
analysis. When D-Orn was employed as a substrate, no NADPH
oxidation or hydroxylated product formation was observed,
indicating that VbsO is specific for the L isomer of Orn (Figure
3B). Both D- and L-lysine were found to be nonsubstrate
effectors of VbsO, as they activated NADPH oxidation, however
no N6-hydroxylysine was detected by analytical HPLC (Figure
S4, Supporting Information). Finally, N5-((R)-3-hydroxybutyryl)-
L- and D-Orn (10) were tested to probe the timing of hydroxy-
lation in the vicibactin biosynthetic pathway as well as VbsO
substrate specificity. Neither compound was found to be a
substrate nor a nonsubstrate effector of VbsO as no hydroxylated
product or NADPH oxidation was observed (Figure S5, Sup-
porting Information).
The hydroxamate substrates D- and L-9 were prepared using
a synthetic scheme based on methodology first illustrated by
Milewska and Chimiak31 and later optimized by Phanstiel.32
As shown in Scheme 2, oxidation of the free amine of N2-Boc-
D- or L-ornithine tert-butyl ester (4) with benzoyl peroxide
afforded the corresponding N5-(benzoyloxy)amines D-5 and L-5
in 36% and 80% yield respectively. Subsequent acylation with
acid chloride 6 under biphasic conditions afforded the fully
protected hydroxamates D- and L-7 in 97% yield for both
compounds. Subsequent removal of the benzoyl (Bz) and acetyl
(Ac) protecting groups by treatment with a 10% NH3/MeOH
solution and of the tert-butyl-carbonate (Boc) and tert-butyl (t-
Bu) groups with trifluoroacetic acid (TFA) provided the TFA
salts of D-9 in 51% overall yield from D-7 and L-9 in quantitative
yield from L-7.
Detailed synthetic protocols and spectroscopic data can be
found in the Supporting Information.
Characterization of VbsA. The VbsA protein exhibits se-
quence similarity to several bacterial acyltransferases11 found
in gene clusters associated with the biosynthesis of hydroxamate
(31) Milewska, M. J.; Chimiak, A. Synthesis 1990, 233–234.
(32) Bergeron, R. J.; Phanstiel, O. I. V. J. Org. Chem. 1992, 57, 7140–
7143.
(33) Thariath, A.; Socha, D.; Valvano, M. A.; Viswanatha, T. J. Bacteriol.
(35) Sokol, P. A.; Darling, P.; Woods, D. E.; Mahenthiralingam, E.; Kooi,
C. Infect. Immun. 1999, 67, 4443–4455.
1993, 175, 589–596.
(34) Pohlmann, V.; Marahiel, M. A. Org. Biomol. Chem. 2008, 6, 1843–
(36) van Berkel, W. J.; Kamerbeek, N. M.; Fraaije, M. W. J. Biotechnol.
2006, 124, 670–689.
1848.
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Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
A R T I C L E S
Figure 3. Characterization of VbsO as an L-ornithine N5-hydroxylase. (A) Schematic of the VbsO-catalyzed conversion of L-Orn to N5-hydroxy-L-ornithine
(L-3) and proposed structure of the Fmoc-derivatized product. ESI-HRMS m/z data of the purified product resulting from incubation of VbsO with L-Orn and
subsequent Fmoc-derivatization is consistent with bisFmoc-N5-hydroxyornithine (bisFmoc-3). (B) HPLC traces (263 nm) showing the Fmoc-derivatized
products resulting from incubation of L- or D-Orn (500 µM) with VbsO (5 µM) in the presence of FAD (50 µM), NADPH (2 mM) and pH 8.0 Tris (50 mM).
Peaks corresponding to bisFmoc-Orn appear at tR 21.9 min and bisFmoc-L-3 at tR 20.8 min. (C) Plot of initial velocity versus L-Orn concentration, with the
best fit curve to the Michaelis-Menten equation. Error bars are drawn at one standard deviation.
containing siderophores. The most notable homologue is IucB,37
a biochemically characterized acetyl-CoA-dependent N6-hy-
droxylysine N6-acetyltransferase involved in the biosynthesis
of the E. coli siderophore aerobactin. We hypothesized that
VbsA catalyzes the transfer of an (R)-3-hydroxybutyryl group
from the CoA thioester 11 to N5-hydroxy-ornthine 3 to form
N5-((R)-3-hydroxybutyryl)-N5-ornithine 9 (Figure 4). Although
genes encoding for the enzymes necessary for the biosynthesis
of thioester 11 are not found in the Vbs cluster, Rhizobium
species are known to utilize 11 for the production of polyhy-
droxybutyrate38 thereby indicating its bioavailability in the cell.
Initial activity assays revealed that incubation of VbsA with
synthetic L-3 and thioester 11 resulted in the formation of two
new peaks in the analytical HPLC trace with retention times of
6.6 and 8.5 min, identical to that of hydroxamate L-9 and
CoASH respectively, which were absent from control experi-
ments in which VbsA was omitted (Figure 4B). The compound
eluting at 6.6 min was isolated and subjected to MS to confirm
its identification as hydroxamate L-9. ESI-HRMS analysis
confirmed the presence of a compound with the molecular
We therefore questioned if VbsA would also recognize D-3 as
the hydroxylamine acceptor. Initial activity assays indicated that
D-3 is also a viable substrate for VbsA and yielded two new
peaks in the analytical HPLC trace with retention times of 6.8
and 8.5 min, identical to that of D-9 and CoASH respectively,
which were absent from control experiments in which VbsA
was omitted (Figure 4B). ESI-HRMS analysis of the peak at
6.8 min further confirmed the product identity as D-9 ([M +
Na]+, m/z calcd: 257.1108, found: 257.1112) (Figure S7,
Supporting Information).
To gain further insight into the substrate specificity of VbsA,
kinetic studies were undertaken with hydroxylamines L- and D-3
by monitoring the rate of CoASH formation using the DTNB
assay. At a fixed thioester 11 concentration of 300 µM, the Km
of VbsA for L-3 was 17.9 ( 2.1 µM and the kcat for the reaction
was 504 ( 12 min-1 (Figure 4C). Under the same assay
conditions, the Km of VbsA for D-3 was 319 ( 47 µM, and the
kcat was 1960 ( 98 min-1. Using the catalytic efficiency criterion
(kcat/Km), L-3 is only a 5-fold better substrate than the D-isomer.
The hydroxylamine promiscuity of VbsA is analogous to that
of IucB, which was shown to fully acetylate both enantiomers
of N6-hydroxylysine as well as N5-hydroxy-L-ornthine 3, N-
methylhydroxylamine and even hydroxylamine. However, Neiland
and co-workers found that IucB39 is selective for hydroxy-
lamines over amines, as Lys was not a substrate. To test if VbsA
would recognize amine substrates as acyl acceptors, VbsA was
incubated with D- or L-Orn and thioester 11 and the formation
+
formula C9H18Na1N2O5 (calcd: 257.1108, found: 257.1109),
consistent with the sodium adduct ion of 9 (Figure S6,
Supporting Information).
At the outset of these studies the timing of C2-epimerization
of the ornithine moiety in the vicibactin scaffold was not known.
(37) Coy, M.; Paw, B. H.; Bindereif, A.; Neilands, J. B. Biochemistry 1986,
25, 2485–2489.
(38) Trainer, M. A.; Charles, T. C. Appl. Microbiol. Biotechnol. 2006, 71,
(39) de Lorenzo, V.; Bindereif, A.; Paw, B. H.; Neilands, J. B. J. Bacteriol.
1986, 165, 570–578.
377–386.
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A R T I C L E S
Heemstra et al.
Figure 4. Characterization of VbsA as a CoA-dependent N5-hydroxyornithine N-((R)-3-hydroxybutryl)- transferase. (A) Schematic of the VbsA-catalyzed
transfer of (R)-3-hydroxybutyryl- from the CoA thioester (11) to N5 of N5-hydroxy-L- or D-ornithine (L- or D-3). ESI-HRMS m/z data of the purified products
are consistent with N5-((R)-3-hydroxybutyryl)-N5-hydroxy-L- and D-ornithine (9) (B) HPLC traces (220 nm) showing products resulting from the incubation
of 11 (1 mM) and L- or D-3 (1.2 mM) with and without VbsA (1 µM) in pH 7.5 Tris (50 mM). Peaks corresponding to L-9 appear at tR 6.6 min, D-9 at tR
6.8 min, CoASH at tR 8.5 min and 11 at tR 9.4 min. (C) Kinetic traces for the VbsA-catalyzed acyltransfer from 11 to L- or D-3, with the best-fit curve to
the Michaelis-Menten equation. Error bars are drawn at one standard deviation.
of CoASH using the DTNB assay was monitored (Figure S8,
Supporting Information). At pH 8.0 with 1 mM substrate,
increase in the absorbance at 412 nm was not observed, which
shows acyl transfer is not occurring and confirms that D- and
L-Orn are not efficient substrates for VbsA.
dependent enzyme,41 is responsible for the C2 L- to D-
configuration change in the Orn scaffold. PLP-dependent
enzymes that have racemase/epimerase activity normally work
on free amino acids rather than aminoacyl thioesters; therefore,
we expected that VbsL would work before VbsS in the
vicibactin biosynthetic pathway. However, the substrate pro-
miscuity of VbsA, shown to acylate both enantiomers of
hydroxylamine 3 (see above), could allow epimerization to occur
before or after hydroxamate formation. Therefore, our initial
assays focused on ascertaining whether N5-hydroxyornithine 3
or N5-((R)-3-hydroxybutyryl)-N5-hydroxyornithine 9 are sub-
strates for VbsL.
Having established that VbsA is selective for hydroxylamines
over amines, we investigated whether the enzyme is capable of
transferring acyl groups from CoA substrates other than 11 to
hydroxyamine L-3. Preliminary activity assays revealed that
incubation of VbsA and hydroxylamine L-3 with ((S)-3-
hydroxybutyryl)-CoA (12), butyryl-CoA (13) and even acetyl-
CoA resulted in the formation of two new peaks in the analytical
HPLC trace with retention times and masses consistent with
the expected hydroxamate product and CoASH (Figure S9,
Supporting Information). The acyl-CoA promiscuity of VbsA
is in contrast to the high selectivity IucB shows for its cognate
acyl-CoA substrate, acetyl-CoA. Neiland and co-workers re-
ported that in activity assays with IucB, propionyl CoA was no
more than 5% as active as acetyl-CoA and isobutyryl-CoA was
not utilized as a substrate.
Initial activity assays indicated that hydroxylamine 3 is not
an efficient substrate for VbsL. Incubation of VbsL (10 µM)
with L- or D-3 and PLP followed by Fmoc-derivatization and
analysis by chiral HPLC (Chiralcel OD-RH) and comparison
to similarly Fmoc-derivatized synthetic standards showed only
a trace amount of epimerization of the amino acids had occurred
after 2 h (Figure S10, Supporting Information). In contrast,
incubation of hydroxamate L-9 with VbsL (0.5 µM) and PLP
followed by Fmoc-derivatization and analysis by chiral HPLC
showed complete racemization had occurred after 1 h (Figure
5B). Racemization was also observed when D-9 was incubated
with VbsL under the same conditions, confirming that both
diastereomers are accepted as substrates for VbsL (Figure 5C).
Characterization of VbsL. One of the notable features of
vicibactin is that the ornithine residue embedded in its scaffold
is in the D-configuration.40 Inspection of the VbsS NRPS module
does not reveal an epimerization domain (see below). Instead,
Carter et al.11 proposed that VbsL, predicted to be a PLP-
(40) Chen, H. P.; Lin, C. F.; Lee, Y. J.; Tsay, S. S.; Wu, S. H. J. Bacteriol.
2000, 182, 2052–2054.
(41) Eliot, A. C.; Kirsch, J. F. Annu. ReV. Biochem. 2004, 73, 383–415.
9
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Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
A R T I C L E S
Figure 5. Characterization of VbsL as a PLP-dependent epimerase. (A) Schematic of the VbsL-catalyzed racemization of N5-((R)-3-hydroxybutyryl)-N5-
hydroxy-L- or D-ornithine (L- or D-9) and proposed structure of the Fmoc-derivatized products (bisFmoc-L- and D-9). (B) Chiral HPLC traces (263 nm)
showing racemization of L-9 (1 mM) resulting from incubation with VbsL (500 nM), PLP (50 µM) and pH 7.75 HEPES (50 mM) followed by derivatization
with Fmoc-Cl. (C) Chiral HPLC traces (263 nm) showing racemization of D-9 (1 mM) resulting from incubation with VbsL (500 nM), PLP (50 µM) and
pH 7.75 HEPES (50 mM) followed by derivatization with Fmoc-Cl. Peaks corresponding to bisFmoc-D-9 appear at tR 5.4 min and bisFmoc-L-9 at tR 7.6 min.
Peak at tR 6.4 min is an artifact of Fmoc-Cl decomposition in aqueous solution.
No racemization activity was detected with enzyme inactivated
by boiling or in the absence of PLP (data not shown).
histidine and the terminal aspartic acid residues have been shown
to be critical for catalytic activity. In VbsS, however, the critical
histidine is replaced with a proline. This mutation suggests that
the C domain is not catalytically active and does not serve a
role in the biosynthesis of vicibactin.
A general mechanism for enzymatic PLP-dependent epimer-
ization/racemization involves the formation of a Schiff-base
between the C2-amino group and PLP. This intermediate
facilitates the deprotonation and reprotonation at the R-position
by conserved residues in the enzyme, resulting in epimerization/
racemization of the substrate. Therefore, assays of VbsL
performed in D2O should result in enzyme-dependent incorpora-
tion of deuterium into the substrate. VbsL was incubated with
L-9 and PLP in D2O for 18 h followed by Fmoc-derivatization.
The resulting bisFmoc diastereomers were separated using chiral
HPLC and the product peaks were collected. The molecular
formula of the compound that coeluted with bisFmoc-L-9 was
found to be C39H37DN2NaO9+ (calcd: 702.2532, found: 702.2539),
as was that of the compound that coeluted with bisFmoc-D-9
(calcd: 702.2532, found: 702.2518) by LC-QTOF-HRMS
analysis, consistent with deuterium incorporation into 9.
The condensation domain in VbsS is followed by an
adenylation (A) domain; such domains are bifunctional catalysts
for both aminoacyl-AMP formation and subsequent loading of
the activated amino acid onto the free thiol of the Ppant group
on the cognate thiolation (T) domain. The specificities of A
domains in NRPS modules can be predicted using an NRPS
code in which the identity of eight amino acids in the A domain
between a conserved aspartate and lysine confer specificity.44,45
However, a careful comparison of the VbsS A domain amino
acid code (DGESSGGMTK) to those in the data bank did not
find any significant homology to A domains of known specific-
ity, and showed that it is significantly different from A domains
known to activate Orn or N5-hydroxyornithine46 (Table S1,
Supporting Information). To determine the substrate specificity
of the VbsS A domain, the classical ATP-pyrophosphate (PPi)
Characterization of VbsS. Translated VbsS has 1331 amino
acid residues and contains four discernible domains by sequence
analysis. The N-terminal domain displays significant sequence
similarity to a condensation (C) domain and contains all seven
signature sequences (C1-C7). The catalytic core of C domains
has the consensus sequence HHXXXD,42,43 in which the second
(43) Keating, T. A.; Miller, D. A.; Walsh, C. T. Biochemistry 2000, 39,
4729–4739.
(44) Challis, G. L.; Ravel, J.; Townsend, C. A. Chem Biol 2000, 7, 211–
224.
(45) Stachelhaus, T.; Mootz, H. D.; Marahiel, M. A. Chem Biol 1999, 6,
493–505.
(42) Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D. Chem ReV 1997, 97,
(46) Dimise, E. J.; Widboom, P. F.; Bruner, S. D. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 15311–15316.
2651–2674.
9
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A R T I C L E S
Heemstra et al.
Characterization of VbsC. The final step in vicibactin
biosynthesis is proposed to be N2-acetylation of the three
ornithine residues in desacetylvicibactin. Based on genetic
knockout studies and bioinformatic analysis, Carter et al.
predicted that VbsC is the acetyltransferase in the Vbs cluster.11
A knockout of VbsC was studied in transconjugants of Para-
coccus denitrificans and led to the generation of a functional
siderophore as judged by the chrome azurol S assay48 (CAS).
However, MS analysis of the supernatant from a culture of P.
denitrificans harboring the VbsC mutant did not reveal a peak
corresponding to vicibactin but instead had a peak of 649.7,
corresponding to desacetylvicibactin (14). Interestingly, VbsL
knockout strains also produced 14, suggesting that VbsC is
specific for the C2-D-configuration in the ornithine moiety.
To investigate the activity and selectivity of VbsC, we first
obtained the C2-D- and L-isomers of desacetylvicibactin 14 from
L. leguminosarum strains with either VbsC or VbsL inactivated11
(generously provided by Prof AWG Johnston). After growing
the strains under iron-deficient conditions, we used preparative
HPLC to separate out and purify the desacetylvicibactin isomers
from the supernatant. Desacetylvicibactin 14 has a distinct UV
profile with an absorbance maximum at 210 nm that aided in
the detection of this siderophore. The identity of the previously
characterized apo-siderophores was confirmed by HRMS and
1H NMR analysis (Figure S11 and S12, Supporting Information);
stereochemistry was determined by saponification of the triester
with LiOH, and chiral HPLC analysis of the resulting monomer
in comparison with diastereomeric standards prepared syntheti-
cally (Figure S13, Supporting Information).
Figure 6. Relative activities of the VbsS adenylation (A) domain with L-
and D-Orn, L- and D-3 and L- and D-9 as assessed by ATP-[32P]PPi exchange
assays.
exchange assay was used to monitor the reversible incorporation
of [32P]PPi into ATP in a substrate-dependent fashion47 (Figure
6). In the presence of 5 mM amino acid, the relative activities
were obtained as follows: D-9 (100%), L-9 (28.6%), L-3 (0.9%),
D-3 (0.04%), L-Orn (0.7%), and D-Orn (0.04%) (Figure 6). This
data clearly shows that hydroxamate D-9 is the preferred
substrate for VbsS; however, the C2-L diastereomer (L-9) is also
recognized to a lesser extent. Interestingly, we find that C-His6-
tagged VbsS, expressed without VbsG, is not active in the ATP-
PPi exchange assay. The role of VbsG in VbsS activity is not
yet clear and is the subject of ongoing investigation.
Protein sequence alignment indicated that the third domain
in VbsS resembles a typical carrier protein or T domain in NRPS
systems with Ser1010 as the proposed site for posttranslational
priming with phosphopantetheine to convert inactive apo T
domains into active holo domains. The purified VbsSG complex
was assayed for covalent attachment of a phosphopantetheinyl
group by Sfp, a phosphopantetheinyltransferase from B. subtilis
with broad specificity. Under the assay conditions, Sfp rapidly
(15 min) modified VbsS with [14C]Ac-CoA as a test substrate
for acyl-pantetheinylation. The stoichiometry of Ppant incor-
poration was estimated to be ∼50% based on the specific
radioactivity of [14C]Ac-CoA and the concentration of VbsS in
the VbsSG pair (Data not shown).
With the C2-D- and L-isomers of desacetylvicibactin in hand
we first incubated purified VbsC with D-14 and acetyl-CoA for
1, 2, 4, 8, 16, and 32 min. The reactions were halted by the
addition of TFA and HPLC analysis revealed the time dependent
appearance of three new peaks in the analytical HPLC chro-
matogram with retention times of 11.0, 11.9, and 12.8 min,
which were absent from control reactions containing VbsC
inactivated by boiling (Figure 7). The compounds with retention
times of 11.0 and 11.9 min were isolated from a large-scale
incubation that was stopped after 8 min. The molecular formula
for the peak at 11.0 min was deduced to be C29H51N6O13+ (calcd:
691.3509, found: 691.3535) by ESI-HRMS analysis, consistent
with the protonated ion of monoacetyled 15 (Figure S14,
Supporting Information). ESI-HRMS analysis of the peak at
+
11.9 min yielded a molecular formula of C31H53N6O14 (calc.:
733.3614, found 733.3622) consistent with the protonated ion
of diacetylated 16 (Figure S15, Supporting Information). The
compound with a retention time of 12.8 min was isolated from
a large-scale reaction that was stopped after 32 min and ESI-
+
HRMS analysis yielded the molecular formula C33H55N6O16
(calcd: 775.3777, found 775.3735) confirming its identity as
protonated vicibactin (VB, Figure S16, Supporting Information).
Although there is a C-terminal thioesterase domain in VbsS
which should be the cyclotrimerization catalyst for D-9 chains
bound to the T domain our assays so far with either VbsS or
the VbsSG complex did not detect release of desacetylvicibactin
(D-14). Detailed assessment of whether VbsS accumulates
tethered linear dimeric and trimeric forms of D-14 on the T
domain and/or the TE domain prior to cyclization to the 30
membered trilactone scaffold in analogy to the action of
enterobactin synthetase will be the subject of a subsequent study.
To further verify that VbsC is indeed catalyzing conversion
of desacetyl-D-vicibactin 14 to vicibactin, an assay containing
VbsC, D-14 and acetyl-CoA was halted after 1 h by the addition
of Fe(ClO)3, which precipitated the enzyme and converted the
product to its corresponding ferric complex. A new peak in the
HPLC trace that coeluted with authentic ferrivicibactin (isolated
from wild type R. etli) and exhibited trihydroxamate-Fe complex
absorption (λ max ) 435 nm) was observed and ESI-MS
analysis indicated a mass consistent with ferri-vicibactin ([M
(47) Santi, D. V.; Webster, R. W., Jr.; Cleland, W. W. Methods Enzymol.
1974, 29, 620–627.
(48) Payne, S. M. Methods Enzymol. 1994, 235, 329–344.
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Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
A R T I C L E S
Figure 7. Characterization of VbsC as a CoA-dependent desacetyl-D-vicibactin N2-acetyltransferase. (A) Schematic of the VbsC-catalyzed, triacetylation of
desacetyl-D-vicibactin (D-14) with 3 equiv of acetyl-CoA and ESI-HRMS m/z data of purified products. (B) HPLC traces (220 nm) showing the time-
dependent appearance of mono-, di- and triacetylated products resulting from the incubation of D-14 (1 mM) and acetyl-CoA (4 mM) with VbsC (10 µM)
in pH 7.75 HEPES (50 mM). Peaks corresponding to CoASH appear at tR 8.1 min, Acetyl-CoA at tR 9.1 min, D-14 at tR 10.2 min, monoacetylated D-15 at
tR 11.0 min, diacetylated D-16 at tR 11.9 min, and VB at tR 12.8 min.
+ H]+ m/z calcd: 828.2835, found: 828.2855) (Figure S17 and
S18, Supporting Information).
products are associated with some aspect of iron transport.
Hydroxamates have the ability to capture ferric iron through a stable
five membered ring chelate and when arranged in a trimer, as in
the siderophore vicibactin, a tight six coordinate species results
(see Figure 1). Trihydroxamates comprise the primary siderophores
essential to the growth of a variety of microorganisms ranging from
pathogens to beneficial agriculture symbionts. Included are the 33-
membered desferrixoamine13 from Streptomycetes, the 36-mem-
bered fusarinine C10 of Aspergillus fungal strains, and the 30-
membered vicibactin from Rhizobia. Each of these macrocycles
is the result of an enzymatic cyclotrimerization of 11, 12, and 10-
atom monomer units, respectively. During the oligomerization-
cyclization reactions involved in the biosynthesis of the cyclic
triesters, a terminal hydroxyl group acts as a nucleophile to capture
a NRPS-bound activated acyl thioester of a separate monomer for
two head to tail esterifications; macrocyclization occurs by in-
tramolecular capture of a TE domain-bound ester. The NRPS-
thiotemplated cyclotrimerization differs from stand-alone macro-
cyclization catalysts (NRPS-independent) that catalyze ATP-
dependent condensation reactions between soluble intermediates,
shown to be operative for amide bond formation and yielding
triamide hydroxamates such as desferrioxamine E. Current data
suggests the NIS pathway14 may have evolved for triamides, while
NRPSs are utilized to generate triester siderophores.
As noted above, VbsC is proposed to be specific for
desacetylvicibactins bearing a C2-D-configured ornithine moiety
owing to the isolation of desacetylvicibactin from VbsL (9-
epimerase) mutants grown in iron-deficient media. To test this
hypothesis, VbsC was incubated with L-14 and acetyl-CoA
under the same conditions as for D-14 and the formation of
CoASH was monitored by UV using the DTNB assay. No
increase in the absorbance at 412 nm (due to the reaction
between the free thiol of CoASH, generated by enzyme-
catalyzed acyltransfer, and DTNB was observed), highlighting
the strong preference of VbsC for D-14 (Figure S19, Supporting
Information). Furthermore, analytical HPLC analysis of reactions
19
halted after 2 h by the addition of either TFA or Fe(ClO)3
indicated that negligible acetylation of L-14 had occurred (data
not shown).
Discussion
Nature seems to have specifically formulated the hydroxamic
acid functionality49 to serve as a site of iron ligation in siderophore
scaffolds. As such, the majority of hydroxamate-containing natural
(49) Neilands, J. B. Science 1967, 156, 1443–1447.
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A R T I C L E S
Heemstra et al.
As part of an effort to understand the origin and role of
N-hydroxylation in the transformation of readily available amino
acids into nonribosomal peptides and related small molecules, we
have investigated the bacterial strategy for trihydroxamate biosyn-
thesis in the context of vicibactin. Through in Vitro characterization
of recombinant vicibactin biosynthetic proteins and comparison of
the enzymatic products with authentic synthetic standards, we have
been able to elucidate the timing and logic of the hydroxamate
monomer synthesis. In Rhizobia, vicibactin is assembled in the
cytoplasm, exported into the rhizosphere, and then reimported as
the Fe-chelate. The ferric-bound complex provides the Fe essential
to the assembly of active endogenous nitrogenases; these enzymes
are involved in maintaining a usable source of nitrogen for
symbiotic host legumes. Microbial siderophores have also been
linked to iron availability in plants; these factors suggest that
siderophore biosynthesis in Rhizobia is a critical component of
productive symbiosis.
Genes for the biosynthesis of vicibactin were identified by
Carter and co-workers in 2002, and reveal the presence of a
key nonribosomal peptide synthetase, VbsS, presumably re-
sponsible for assembly of the trimer, and several tailoring
enzymes that convert L-Orn to the functionalized hydroxamate
monomer 9. Genetic studies indicate that, in addition to VbsS,
VbsA,G, and O, encoding an acyltransferase, an MbtH-like
protein,50 and an N-hydroxylase, respectively, are essential for
vicibactin production. In contrast, insertional mutants of VbsC
(encoding an acyltransferase) and VbsL (an epimerase) produce
desacetylated form(s) of vicibactin. Bioinformatic analysis
suggests that the 10-atom monomer chain is derived from L-Orn,
but does not reveal the sequence of modification events.
Although tailoring of L-Orn could occur in trans while the
substrate is tethered to the T domain of VbsS, as suggested by
Carter, an alternative pathway, detailed here, involves complete
elaboration of the desacetyl-hydroxamate monomer before
utilization by the VbsS adenylation domain.
Previous studies of siderophore biosynthesis support our
findings that VbsS activates functionalized Orn. For example,
early investigation of the fusigen synthetase, analyzed in cell
free extracts, indicated the direct utilization of a fully function-
alized hydroxamate monomer.51 A key differentiating feature
of fungal and bacterial hydroxamate siderophores is the con-
figuration of the Orn residue (D vs L). It has not been determined
why this difference is maintained, but one objective of our study
was to determine the gate-keeping mechanism that shuttles only
the D-isomer through the vicibactin assembly line.
We began our investigation with VbsO, a flavin-dependent
hydroxylase able to generate reduced flavin with NADPH
without the need for an external reductase, and found that it is
site- and stereospecific for the distal nitrogen in L-Orn. The
intermediate peroxy-flavin species that serves as the active
hydroxylation catalyst requires nucleophilic attack for oxygen
transfer. This mechanism is supported by the fact that while
the N5-amino group of L-Orn is a substrate for N-hydroxylation,
the less nucleophilic N5-acyl-derivative 10 of either D or L-Orn
is unreactive.
necessary gatekeeping function to ensure that the key hydrox-
amate functionality required for iron coordination is assembled.
The fact that VbsA is promiscuous and accepts both the L and
D configuration of hydroxylamine 3 opens the question of when
the C2-position of the Orn residue is epimerized. This issue has
been resolved as noted below. In the utilization of thioester 11,
available from primary fatty acid metabolism, VbsA also installs
the essential hydroxy group that enables downstream head to
tail trimerization of the monomer.
The stereochemistry at the C2-position in each monomer unit
is essential for the formation of a well-defined Fe hexadentate
chelation, and dictates the chirality of the resulting complex.52
As fungal siderophores are predominantly of the L-configuration,
the use of a D-Orn derivative by Rhizobia could be a strategy
to protect the metabolic investment in vicibactin biosynthesis
by limiting the use of ferri-vicibactin by other microorganisms
in the rhizosphere. A general mechanism in secondary metabo-
lism for the formation of D-amino acid residues involves enzyme
catalyzed exchange of the R-proton, rendered acidic by either
formation of a thioester (a covalently tethered substrate to an
NRPS module), or a PLP-derived Schiff base of a soluble
substrate. Bioinformatic analysis of the VbsS NRPS module
does not reveal an epimerization domain, and therefore suggests
that formation of the D-Orn isomer occurs prior to trimerization.
In Vitro studies of VbsL, a PLP-dependent epimerase, cor-
roborates the hypothesis that the preferred substrate is the free
hydroxamate 9. VbsL is highly selective for 9, and does not
efficiently process either L-Orn, or hydroxylamine 3, despite
the availability in each of the requisite free amine for Schiff
base formation. The selection of the hydroxamate monomer by
VbsL also explains why VbsA does not need to maintain
selectivity for the stereochemistry at the C2-position of the
hydroxylamine as only the L-isomer is available for acylation.
We have therefore shown that VbsO, A, and L are necessary
and sufficient to generate the desacetyl-hydroxamate moiety
embedded in the vicibactin trimeric structure.
Along with several tailoring enzymes, the vicibactin gene cluster
also encodes the NRP synthetase VbsS. While biochemical
characterization of VbsO, A, L has elucidated the origin of the
densely functionalized 10 atom monomer, analysis of VbsS would
further substantiate the postulated trimerization mechanism. The
147 kDa VbsS was originally difficult to obtain in purified form
through Ni-affinity chromatography of the His6-tagged construct;
however, it was possible to achieve purification by ammonium
sulfate fractionization and size exclusion column chromatography
of heterologously expressed VbsGS. Coexpression of VbsS with
the MbtH-like protein50 VbsG provided increased amounts of the
active synthetase in the purified protein fraction for reasons that
are not yet understood. Although their function has not yet been
elucidated, small 7-8 kDa MbtH-like proteins are cotranscribed
in many NRPS-encoding gene clusters and have been shown to
be important for metabolite assembly in ViVo.50 The role of VbsG
in improved solubility, stability, or activity of VbsS is the subject
of ongoing investigation.
While the N-terminal C (condensation) domain of VbsS is
presumably not functional based on sequence analysis, it was
possible to assay the first catalytic step performed by the
synthetase A domain. Analysis of the ATP-dependent conver-
sion of the substrate carboxylic acid into the aminoacyl-AMP
ester in the presence of VbsS was accomplished by measuring
the incorporation of 32PPi in ATP as a result of reversible
VbsA is homologous to CoA-dependent N-acyl transferases;
based on VbsO data, we suspected that VbsA acts on a soluble
hydroxylamine, rather that the primary amine. Our results show
N5-hydroxyornithine 3 and not Orn is selectively acylated - a
(50) Lautru, S.; Oves-Costales, D.; Pernodet, J. L.; Challis, G. L. Micro-
biology 2007, 153, 1405–1412.
(51) Anke, H.; Anke, T.; Diekmann, H. FEBS Lett. 1973, 36, 323–325.
(52) Drechsel, H.; Jung, G. J. Pept. Sci. 1998, 4, 147–181.
9
15328 J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009

Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
A R T I C L E S
Figure 8. Logic of vicibactin biosynthesis involves complete functionalization of the Orn-based monomer prior to VbsS catalyzed desacetylvicibactin
assembly. Selection for the C2-D hydroxamate (D-9) monomer is maintained by the adenylation (A) domain of VbsS. Tailoring catalyzed by VbsC leads to
the fully functionalized vicibactin siderophore scaffold. (NOH ) amine N-hydroxylase, AT ) acyltransferase, E ) epimerase)
substrate activation. The use of several candidate Orn-derived
monomers in this ATP-PPi exchange experiment indicated the
hydroxylamines L- and D-3 (and L- and D-Orn) are not accepted
as substrates, and that, while the D-9 diastereomer is preferred,
the L-9 diastereomer is activated to a lesser extent. While we
have not yet succeeded in reconstitution of the cyclotrimerization
in Vitro, our results indicate that the incomplete selectivity of
the VbsS domain for D- vs L-9 activation is consistent with
detection of both D- and L-desacetylvicibactin isomers in mutant
strain noted below.
coelicolor, ligation of N-hydroxycadaverine with a succinyl group
from the Krebs cycle metabolite succinyl-CoA is promoted by an
acyltransferase belonging to the GNAT family.13 Coelichelin
biosynthesis in Streptomyces coelicolor involves hydroxylamine
formylation catalyzed by the formyl transferase CchA in the
presence of N10-formyltetrahydrofoloate.54 In the case of vicibactin,
we find the acyltransferase VbsA couples a ((R)-3-hydroxybutyryl)-
CoA 11 from lipid metabolism with N5-hydroxyornithine 3. While
there are a variety of mechanisms for acylation, flavin dependent
N-hydroxylases are commonly the source of the hydroxyl amine;36
a majority act on aliphatic (Orn, Lys, histamine, cadaverine), and
therefore nucleophilic, amine substrates. Mycobactin biosynthesis
is a single example where a flavoenzyme-mediated hydroxylation
is suggested to follow amine acylation.55 On the one hand this
order of acylation then hydroxylation would avoid formation of
the potentially unstable hydroxylamines; however this sequence
requires the amide to be sufficiently nucleophilic for N-hydroxy-
lation by the peroxy-flavin intermediate. Given the essential role
of flavin-monooxygenases in the biosynthesis of virulence-associ-
ated siderophores such as mycobactin55 from Mycobacterium
tuberculosis and pyoverdine56 from Pseudomonas aeruginosa,
further structural and functional analysis could be of value for the
development and identification of new therapeutic targets.57
Genetic knockouts in the vicibactin gene cluster, prepared
and studied in transconjugants of Paracoccus denitrificans by
Carter and co-workers,11 further address the issue of stereose-
lection in vicibactin biosynthesis and point to the secondary
gatekeeping function of the last-acting acetyltransferase VbsC.
While the VbsC mutant makes desacetylvicibactin as expected,
the VbsL knockout also produces a functional siderophore of
the same molecular weight, presumed to be the L-isomer of
desacetylvicibactin 14. These data suggests that VbsL and VbsC
each act at different stages of assembly to ensure only the trimer
of the D-ornithine containing monomer is elaborated to mature
vicibactin. In order to confirm the stereoselectivity conferred
by VbsC, we isolated two samples of desacetylvicibactin 14
from the VbsL and VbsC knockouts and tested each in Vitro as
substrates for purified VbsC. Confirmation of the suggested
stereochemistry of the starting material trimers was accom-
plished by saponification and comparison of the resulting
monomer with synthetic D and L-hydroxamate 9 by chiral HPLC.
Corroborating the in ViVo work, purified VbsC only accepts the
trimer of the D-hydroxamate 9 for acetyltransfer with acetyl-
CoA to produce mature vicibactin, identified following treatment
with Fe by coelution with an authentic sample of ferrivicibactin
isolated from R. etli CFN42 and HRMS. The purification of
ferrivicibactin from R. etli CFN42 is also noteworthy as it
confirms our initial assumption that vicibactin is a native
siderophore of this strain.
Acknowledgment. This work was supported by an NIH grant
to C.T.W. (AI042738) and by an NIH NRSA fellowship to J.R.H
(F32GM083464); Elizabeth S. Sattely is a Damon Runyon Fellow
supported by the Damon Runyon Cancer Research Foundation
(DRG-1980-08). We thank Prof. Andrew W. B. Johnston for
provision of R. leguminosarum strains J372 and J369 and Prof.
Dewey G. McCafferty and Amanda Jane Hoertz for helpful
suggestions regarding the purification of VbsS.
Supporting Information Available: Figures S1-S19, Table
S1, detailed experimental procedures, and spectroscopic data
for the preparation of compounds 3 and 9. This material is
available free of charge via the Internet at http://pubs.acs.org.
Hydroxamates are a common motif in bacterial siderophore
scaffolds, and a prevalent mechanism for iron capture in fungi.
Several strategies have coevolved for hydroxylamine acylation to
produce the ubiquitous hydroxamate with distinct acyl moieties.
In Pseudomonas, an NRPS C domain catalyzes acylation of
phosphopantetheinyl-tethered oxazolinyl acyl moiety with soluble
N-hydroxy-histamine to generate prepseudomonine in the chain
release step.53 For desferrioxamine E production in Streptomyces
JA9056008
(54) Challis, G. L. Microbiology 2008, 154, 1555–1569.
(55) Krithika, R.; Marathe, U.; Saxena, P.; Ansari, M. Z.; Mohanty, D.;
Gokhale, R. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2069–2074.
(56) Visca, P.; Imperi, F.; Lamont, I. L. Trends Microbiol. 2007, 15, 22–
30.
(57) Miller, M. J.; Zhu, H.; Xu, Y.; Wu, C.; Walz, A. J.; Vergne, A.;
Roosenberg, J. M.; Moraski, G.; Minnick, A. A.; McKee-Dolence, J.;
Hu, J.; Fennell, K.; Kurt Dolence, E.; Dong, L.; Franzblau, S.; Malouin,
F.; Mollmann, U. Biometals 2009, 22, 61–75.
(53) Sattely, E. S.; Walsh, C. T. J. Am. Chem. Soc. 2008, 130, 12282–
12284.
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