Formation of the chromophore of the pyoverdine siderophores by an oxidative cascade

Article abstract of DOI:10.1021/ol034531e

(Matrix pesented) The pyoverdine chromophore is formed in a reaction involving a two-electron oxidation, a conjugate addition, and a second two-electron oxidation. This oxidative cascade can be carried out with polyphenol oxidase (PPO), MnO₂, and cell-free extracts from Pseudomonas aeruginosa.

Full text of DOI:10.1021/ol034531e

ORGANIC
LETTERS
2003
Vol. 5, No. 13
2215-2217
Formation of the Chromophore of the
Pyoverdine Siderophores by an
Oxidative Cascade
,†
Pieter C. Dorrestein,† Keith Poole,‡ and Tadhg P. Begley*
Department of Chemistry and Chemical Biology, 120 Baker Laboratory,
Cornell UniVersity, Ithaca, New York 14853, and Department of Microbiology
and Immunology, Queen’s UniVersity, Kingston, Ontario, Canada K7L 3N6
tpb2@cornell.edu
Received March 26, 2003
ABSTRACT
The pyoverdine chromophore is formed in a reaction involving a two-electron oxidation, a conjugate addition, and a second two-electron
oxidation. This oxidative cascade can be carried out with polyphenol oxidase (PPO), MnO , and cell-free extracts from Pseudomonas aeruginosa.
2
Pathogenic pseudomonas aeruginosa and related Pseudomo-
nads secrete a pyoverdine siderophore1 (1) under iron
starvation conditions (Figure 1).2 This siderophore is a
virulence factor that functions by complexing Fe3+ prior to
transport into the cytoplasm via the transmembrane protein,
FpvA.3 Pyoverdines are species-specific peptides consisting
of 6-19 amino acid residues, containing D-amino acids, one
or more hydroxamates, and the characteristic pyoverdine
chromophore.4
Incorporation studies have demonstrated that the pyover-
dine chromophore is derived from tyrosine (2)5 and L-2,4-
diaminobutyric acid (3).6 It has also been demonstrated that
the pVc gene cluster is essential for the biosynthesis of the
Figure 1. Structure of a pyoverdine. In subsequent structures, the
peptide and the bisamide substituents on the chromophore are
chromophore (Scheme 1).7
represented as R groups.
† Cornell University.
‡ Queen’s University.
(1) Pseudobactin and fluorescein are two altenative names used for
pyoverdine, particularly in the older literature.
Several compounds (4-8, Figure 2), which are related to
(2) Budzikiewicz, H. FEMS Microbiol. ReV. 1993, 104, 209-228.
(3) Schalk, I. J.; Kyslik, P.; Prome, D.; Dorsselaer A. V.; Poole, K.;
Abdallah, M. A.; Pattus, F. Biochemistry 1999, 38, 9357-9365.
(4) Fuchs, R.; Budzikiewicz, H. Curr. Org. Chem. 2001, 5, 265-288.
(5) Thompson, B. N.; Gould S. J. Tetrahedron 1994, 50 (33), 9865-
9872.
the pyoverdines, have been isolated from various Pseudomo-
nads. Of these, it has been demonstrated that 4 is a precursor
(6) Bo¨ckmann, M.; Taraz, K. Budzikiewicz., Z. Naturforsch., C: Biosci.
1997, 52, 319-324.
10.1021/ol034531e CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/03/2003

addition would give 12. A facile tautomerization reaction
would then reestablish the catechol functionality in 13 and
set up the system for a second two-electron oxidation to give
o-quinone 14. A sequence of two tautomerizations yields 16.
Analogous oxidative cascades have been observed for the
phenoxazinone synthase catalyzed formation of the phenox-
azinone chromophore in actinomycin biosynthesis,17 the
polyphenol oxidase catalyzed formation of 5,6-dihydroxy-
indole in melanin biosynthesis18 and in the biomimetic
synthesis of styelsamine.19
Scheme 1. Precursors to the Pyoverdine Chromophore
To test this mechanistic proposal, phenol 20, the simplest
analogue of 9, was synthesized20 and incubated with polyphe-
nol oxidase. Polyphenol oxidase is an enzyme known to
hydroxylate a variety of phenols to the corresponding
catechols and also to oxidize catechols to the corresponding
o-quinones (eq 1).21
Figure 2. Isolates that are biogenically related to pyoverdine.
Two major products were formed in this reaction, which
were characterized by 1H NMR, MS, UV-vis, and fluores-
cence spectroscopy. These were shown to be structures 21
and 22.22 Polyphenol oxidase catalyzed oxidation of catechol
23 also gave a mixture of 21 and 22 supporting the
to pyoverdine,8 that 5 is readily oxidized to pyoverdine in a
nonenzymatic reaction,9 and that 8 is secreted by a pyover-
dine mutant overexpressing the pVc gene cluster.10 Several
mechanistic proposals for the formation of the chromophore
have been described, based on isolation studies,11,12 labeling
studies,5 and mutant complementation.13
We suggest a new mechanistic proposal for the formation
of the pyoverdine chromophore that is consistent with the
formation of 4-8 by related chemistry (Figure 3).14-16 In
(8) Hohlneicher U.; Scha¨fer, M.; Fuchs, R.; Budzikiewicz, H. Z.
Naturforsch. 2001, 56C, 308-310.
(9) Teintze, M.; Leong, J. Biochemistry 1981, 20, 6457-6462.
(10) Stintzi, A.; Cornelis, P.; Hohnadel, D.; Meyer, J. M.; Dean, C.;
Poole, K.; Kourambas, S.; Krishnapillai, V. Microbiology 1996, 142, 1181-
1190.
(11) Jacques, P.; Onega, M.; Gwose, I. Seinsche, D.; Schro¨der, H.;
Delfosse, P.; Thonart, P.; Taraz, K.; Budzikiewicz, H. Z. Naturforsch. 1995,
50C, 622-629.
(12) Longerrich, I.; Taraz, K.; Budzikiewics, H.; Tsai, L.; Meyer, J. M.
Z. Naturforsch. C. Biosci. 1993, 48, 425-429.
(13) Maksimova, N. P.; Blazhevich, O. V.; Formichev, Y. K. Mol. Genet.,
Mikrobiol. Virusol. 1993, 5, 22-26.
(14) Teintze, M.; Leong, J. Biochemistry 1981, 20, 6457-6462.
(15) Michalke, R.; Taraz, K.; Budzikiewicz, H.; Thonart, P.; Jacques,
P. Z. Naturforsch. 1997, 52C, 855-857.
(16) Hohlneiger, U.; Hartmann, R.; Taraz, K.; Budzikiewicz, H. Z.
Naturforsch. 1995, 50C, 337-344.
(17) Barry, C. E.; Nayar, P. G.; Begley T. P. Biochemistry 1989, 28,
6323-6333.
(18) Fatibello-Filho, O.; Viera, I. C. Analyst 1997, 122, 345-350.
(19) Skyler, D.; Heathcock, C. H. Org. Lett. 2001, 3, 4323-4324.
(20) See the Supporting Information.
(21) Kazandjian, R. Z.; Klibanov, A. M. J. Am. Chem. Soc. 1985, 107,
5448-5450.
(22) A solution of 3 mg of 20 in 15 mL of 40 mM KPi at pH 6.8 was
incubated with polyphenol oxidase (40 µg) until maximum fluorescence
was observed. At this point, the reaction was quenched with methanol and
filtered through a membrane by centrifugation (5000 Da cutoff) to remove
the precipitated protein. The methanol was removed under reduced pressure
followed by removal of the water by lyophilization. The fluorescent product
was purified by cation exchange chromatography (Amberlite CG-50, 6 g)
using a gradient of 0.001 M HCl to 0.2 M HCl and all the fluorescent
fractions were lyophilized to give 22. (200 µg, 6%) as a green solid: 1H
NMR (400 MHz, D2O) 7.62 (d, J ) 8.4, 1H), 6.99 (s, 1H), 6.97 (s, 1H),
6.50 (d, J ) 9.6, 1H), 4.05 (br t, 2H), 3.32 (br t, 2H), 2.05 (br m, 2H);
fluorescence em (1:1 MeOH/H2O, pH 7.5, ex 390, λmax) 445; UV-vis (1:1
MeOH/H2O, λ) pH 9.5; 406 (ꢀ ) 1 × 105), 235, 213, pH 7.5; 391, 265,
233, pH 3.5; 359, 308, 248, 220. MS ESI (m/z, M + H+) 217; daughter ion
217 (MS MS, m/z, M+) 217, 189 (retro Diels-Alder), 160 (loss of nitrile).
A second, light green fluorescent fraction was isolated which corresponded
to 21 (see the Supporting Information). Incubation of 21 with polyphenol
oxidase gives 22.
Figure 3. Mechanistic proposal for the formation of the pyoverdine
chromophore.
this proposal, hydroxylation of 9 would give catechol 10.
Oxidation of 10 to the o-quinone 11 followed by a conjugate
(7) Stintzi, A.; Johnson, Z.; Stonehouse, M.; Ochsner U.; Meyer, J. M.;
Vasil, M. L.; Poole, K. J. Bacteriol. 1999, 181, 4118-4124.
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Org. Lett., Vol. 5, No. 13, 2003

intermediacy of this compound. In addition, polyphenol
oxidase catalyzed the oxidation of 21 to 22. Products 21 and
22 were also obtained by oxidizing 23 with manganese
dioxide.23 The formation of the chromophore 22, by the
polyphenol oxidase catalyzed oxidation of 20 and 23, as well
as the isolation of 21 supports the proposed oxidative cascade
for the formation of the pyoverdine chromophore (Scheme
2).
Scheme 2
Figure 4. Oxidation of 20 to 22 by cell free extract from
Pseudomonas aeruginosa: (1) 20 and cell-free extract from cells
grown in the absence of Fe(III); (2) same as 1, without cell-free
extract; (3) same as 1 except that the cell free extract was boiled;
(4) 20 and cell-free extract grown in the presence of 40 µM
Fe(III); (5) 20 and cell-free extract from pvdS- cells grown in the
absence of Fe(III); (6) 20 and cell-free extract from pvdS- cells
grown in the presence of 40 µM Fe(III). The assay procedure is
described in the Supporting Information. Each activity is an average
of four independent experiments.
To test 20 and 23 as possible substrates for the pyoverdine
biosynthetic enzymes, these compounds were treated with
cell free extract from P. aeruginosa, grown under iron
limiting conditions to induce the pyoverdin biosynthetic
genes. Under these conditions, 20 and 23 were oxidized to
22 (Figure 4) by cell-free extract. The oxidation of 23 to 22
was 5 times faster than the oxidation of 20 to 22. The activity
was not detected when the cell-free extract was omitted and
reduced to 4% when the cell-free extract was boiled. To
further eliminate the possibility that the oxidation was
mediated by free transition metal ions in the cell-free extract,
the assay was repeated using cell-free extract from cells
grown in the presence of 40 µM Fe(III), conditions known
to repress expression of the pyoverdine biosynthetic genes.7
Under these conditions, the production of 22 was decreased
by at least a factor of 330.24 Finally, cell-free extract prepared
from a strain mutated in an essential transcriptional regulator
(the sigma factor pVdS) for pyoverdine biosynthesis25 did
not catalyze the oxidation of 20 to 22, even when the medium
did not contain Fe(III). This is the first direct confirmation
that the chromophore producing enzymes are regulated by
PvdS. These observations also confirm that the highly
truncated version of 9 is a substrate for the enzyme(s)
involved in the biosynthesis of the pyoverdine chromophore.
Acknowledgment. We thank Cynthia Kinsland for bring-
ing this system to their attention and Iain Lamont for
providing the pvdS mutant. This research was supported by
a grant from the Petroleum Research Fund.
Supporting Information Available: Procedures for the
synthesis and chemical and enzymatic oxidation of 20, 21,
and 23. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL034531E
(23) Hirano, M.; Yakabe, S.; Chikamori, H.; Clark, J. H.; Morimoto, T.
J. Chem. Res., Synop. 1998, 770-771.
(24) The activity was observed without any additional cofactors.
(25) Ochsner, U. A.; Johnson, Z; Lamont, I. L.; Cunliffe, Heather E.;
Vasil, Michael L. Mol. Microbiol. 1996, 21, 1019-1028.
Org. Lett., Vol. 5, No. 13, 2003
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