Structural and functional analysis of pantocin A: An antibiotic from Pantoea agglomerans discovered by heterologous expression of cloned genes

Article abstract of DOI:10.1002/anie.200351053

Small-molecule chemistry and biochemical techniques have been used to determine the structure and mode of function of pantocin A (see structure), a naturally occurring antibiotic. Pantocin A functions as an inhibitor of histidine biosynthesis, as shown by its ability to inhibit the action of L-histidinol phosphate aminotransferase, which converts imidazole acetol phosphate to L-histidinol phosphate in Escherichia coli.

Full text of DOI:10.1002/anie.200351053

Communications
Antibiotic Structure Determination
Structural and Functional Analysis of Pantocin A:
An Antibiotic from Pantoea agglomerans
Discovered by Heterologous Expression of
Cloned Genes**
Mi Jin, Liang Liu, Sandra A. I. Wright, Steven V. Beer,
and Jon Clardy*
The epiphytic bacterium Erwinia amylovora causes fire
blight, a devastating disease of rosaceous plants such as
apple and pear.[1] The closely related but nonpathogenic
bacterium Pantoea agglomerans (formerly E. herbicola),
which usually accompanies E. amylovora in the wild,[2]
produces a family of antibiotics that inhibit E. amylovora in
a poorly understood strain-dependent fashion.[3,4] The numer-
ous members of this antibiotic family constitute a source of
biocontrol agents for fire blight, a pool of new and potentially
useful antibiotics against human bacterial pathogens, and a
model of nature's ability to produce a “library” of biologically
active molecules. The complexity of antibiotic production,
especially the multiple antibiotics produced by a given strain
and low production in liquid culture, has hampered chemical
investigations. We have taken a genomic approach to
deconvolute the multiple antibiotics and to address the
production problems by a particular strain. Heterologous
expression of a genomic DNA library from P. agglomerans in
Escherichia coli provides access to the small-molecule anti-
biotics. Since both P. agglomerans and E. coli are members of
the widely distributed Enterobacteriacea, E. coli should be a
suitable host for heterologous expression of P. agglomerans
genes that encode antibiotics. Herein we report the isolation
and structure determination of pantocin A, a new peptide-
derived antibiotic, and its molecular target.
A genomic library of P. agglomerans strain Eh318 was
constructed in E. coli strain DH5a.[5] Two distinct antibiotic-
[*] Prof. J. Clardy,++ M. Jin, L. Liu
Department of Chemistry and Chemical Biology
Cornell University
Ithaca, NY 14853-1301 (USA)
E-mail: jon_clardy@hms.harvard.edu
Prof. S. A. I. Wright,+ Prof. S. V. Beer
Department of Plant Pathology
Cornell University
Ithaca, NY 14853 (USA)
[þ] Current address:
Plant Pathology and Biocontrol Unit
SLU, 750 07 Uppsala (Sweden)
[
þþ] Current address:
Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School
Boston, MA (USA)
Fax: (+1)617-432-3702
[**] This work was supported by the National Institutes of Health
(National Cancer Institute, R01-CA24487).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2898
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DOI: 10.1002/anie.200351053
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Angewandte
Chemie
Table 1: NMR data for 2.
producing cosmid clones, pCPP702 and pCPP719, were
identified, and the two antibiotics they produced were
trivially named pantocin A and B, respectively. One indica-
tion that the two antibiotics were different came from studies
which showed that arginine suppresses the antibiotic activity
of pantocin B while histidine suppresses the antibiotic activity
of pantocin A. In previous work, pantocin B (PB, 1) was
structurally characterized, and its ability to potently inhibit N-
Atom No.
13C[a]
1H[b]
15N[c]
1
2
3
126.3
131.5
66.4
6.35 (1H, dd, 6.0, 2.0)
6.15 (1H, dd, 6.0, 1.7)
5.21 (1H, brs)
4-N
145.1 (s)
36.5 (s)
5
6
167.9
50.2
3.58 (1H, dd, 13.6, 7.2)
6-NH2
7
28.6
2.57 (1H, m)
2.33 (1H, m)
5.12 (1H, dd, 6.0, 2.4)
8
5
1
9
8
9
1’
95.5
143.3
166.2
2
4
6
N
CH3
3
H
N
H2N
H
CO2H
N
1'
2’-NH
3’
4’
8.27 (1H, d, 7.5)
4.29 (1H, m)
2.50 (1H, dd, 15.2, 7.0)
2.39 (1H, dd, 15.2, 5.7)
126.0 (d, 93.0)
113.6 (t, 90.0)
O
NH2
O
H2N
49.8
37.9
HN
2'
O
SO2CH3
O
OH
3'
O
5’
5’-NH2
6’
171.6
O
1 (PB)
2 (PA)
6.84, 7.72 (2H, brs)
172.6
[a] 13C NMR (100 MHz, 258C, [D6]DMSO). [b] 1H NMR (500 MHz, 258C,
[D6]DMSO) assignments based on 1H-13C HMQC. [c] 15N NMR
(40.5 MHz, 258C, D2O). The 15N chemical shift was calibrated indirectly
based on the gyromagnetic ratio of 15N and 1H according to refer-
ence [12].
acetylornithine transaminase, which catalyzes a committed
step in arginine biosynthesis, was established.[6–8] The charac-
terization of pantocin A (PA, 2) was plagued by inconsistent
and low levels of production in liquid culture, together with its
acid, base, and thermal lability. Significant reproducible
production was eventually achieved by culturing E. coli
XL2Blue harboring a 3.5 kb subclone of pCPP702 in the
high copy number plasmid pUC19 (pUC449).
Isolation of 2 was guided by antibiotic activity against the
E. amylovora strain Ea273(IC 50 = 200 nm). Pantocin A (2)
was purified from large-scale fermentation of E. coli
XL2Blue-pUC449 by four chromatographic separations
using anion-exchange, reverse-phase, and hydrophilic inter-
actions. This compound is an optically active small molecule,
and its structure was established by spectroscopic methods
and synthesis of a hydrogenated derivative.
its HMBC correlations with H6 and H7 required an amino
group at C6, thus completing the planar structure of 2.
The stereocenters at C3’, C3, and C6 were defined by the
hydrogenation of 2, NOESY experiments, and synthesis.
Hydrogenation of 2 gave 3 as a single diastereomer, and
NOESY correlations between H3/H9 and H6/H9 require H3
and H6 to be on the same face (Scheme 1). Hydrolysis of 3
gave 4 and l-aspartic acid (5), which set the absolute
configuration at C3’ as S. Both 3 and its 3(R),6(R),9(R) dias-
tereomer were prepared from 6, which was synthesized from
PA (2) has a molecular formula of C13H16N4O5 as
determined from HRFAB-MS studies. The 13C NMR
(Table 1) and DEPT spectra indicate four carbonyl, four
olefinic, three methine, and two methylene carbon atoms.
Three vinylic, a highly deshielded methine, two Ca amino
acid, four Cb amino acid, and three exchangeable protons
could be characterized from 1H and 1H-13C HMQC NMR
spectra recorded in D2O and [D6]DMSO. A loss of m/z = 132
in the tandem ESI-MS study suggested the presence of an
asparagine residue, which was confirmed by 2D NMR studies.
Two spin systems (H6/H7a,b/H8 and H1/H2/H3) identified by
COSY experiments coupled with HMBC correlations (H6/
C5, H8/C9, H1/C9, and H3/C1’) established a continuous
nine-carbon chain (C5 to C1’) containing the non-asparagine
carbon atoms in 2. Two additional HMBC correlations (2’NH/
C1’ and H3’/C1’) showed that the C9 and asparagine fragments
must be connected by an amide bond.
H
NH2
1
5
HO2C
9
N
CO2H
3'
3
H2N
6
H+
H2
+
CO2H
O
O
2
HN
NH2
H
Pd/C
H2O
OH
H2N
6
9
HN
O
3'
3
O
4
3
CO2H
d, e
O
H
1
CO2CH3
CO2CH3
NHPhF
a, b, c
9
N
3
H3CO2C
BocHN
6
O
O
HO
NHPhF
15N NMR experiments on 15N-labeled 2, which was made
using 15N ammonium sulfate in minimal culture media,
revealed the nitrogen connectivity. A highly downfield shifted
15N resonance (d = 145.1 ppm) with correlations to H1, H2,
H3, H6, and H8 required N4 to be connected to C3, C5, and
C9. A 15N resonance at d = 36.5 ppm, typical of an amine, and
6
7
Scheme 1. a) NaOH/dioxane; CH3I/K2CO3/DMSO; b) H2/Pd-C;
c) (Boc)2O/TEA/DMF; NaOH/CH3OH-H2O; d) H-Asn-OtBu·HCl, EDC/
NMM/DMF; e) TFA/CH2Cl2. Boc=tert-butoxycarbonyl, TEA=triethyl-
amine, EDC=3-(3-dimethylaminopropyl)-1-ethylcarbodiimide,
NMM=N-methylmorpholine, TFA=trifluoroacetic acid.
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l- or d-Glu(OMe)-OMe following a literature procedure[9]
along a biosynthetic pathway has limited applicability in
histidine biosynthesis since many of the intermediates are
ionized phosphate esters that have poor cellular access. Using
the well-characterized histidine biosynthetic enzymes of
E. coli, the step catalyzed by l-histidinol phosphate amino-
transferase, which converts imidazole acetol phosphate to l-
histidinol phosphate [Eq. (1)], was identified as the target of
2.[11]
(Scheme 1). HPLC analysis of synthetic 3 and 3 derived from
2 indicated that the absolute configuration of 3 was 3(S),6(S).
Thus, C3, C6, and C3’ all have the absolute configuration
corresponding to an l-amino acid, and the absolute stereo-
structure of 2 is as shown.
Perhaps the most striking feature of 2, which to the best of
our knowledge has no precedents in reported natural or
synthetic compounds, is its very existence
given the apparent ease with which it could
L-histidinol phosphate
aromatize to a pyrrole. Removal of a proton
from C3, which is a to the carbonyl group at
C1’, followed by reprotonation at C8 would
generate a pyrrole, and this process likely
underlies the lability of 2. A minor inactive
compound was also recovered on isolating
O
N
aminotransferase
NH2
N
2–
HN
2–
HN
(1)
OPO3
OPO3
L-glutamate
α-ketoglutarate
2, and this was characterized as 8. Compound 8 arises from the
aromatization and hydrolysis of the bicyclic amide bond. No
l-histidinol phosphate aminotransferase is a pyridoxal-5’-
phosphate (PLP) dependent enzyme that couples the reduc-
tive amination of imidazole acetol phosphate with the
conversion of l-glutamate into a-ketoglutarate.
Pantocin A and B share a close functional relationship
that is not apparent from their structures. Both gain access to
their cells via the tripeptide transporter and inhibit trans-
aminase-catalyzed amino acid biosynthetic steps that intro-
duce nitrogen functionality.[6,11] Whereas 1 inhibits arginine
biosynthesis, 2 inhibits histidine biosynthesis. This functional
similarity is unsurprising since they have the common goal of
suppressing the growth of bacterial competitors in a nitrogen-
poor environment—an environment where import by tripep-
tide transporters and amino acid biosynthesis are essential.
The structural and functional analysis of 2 (PA) reported
here used techniques of small-molecule chemistry and
biochemical analysis. The cosmid library/heterologous
expression approach provides ready access to the DNA
conferring production of 2 on E. coli XL2Blue-pUC449.
Analysis of this DNA provides insights into the biosynthesis
of 2 and identifies strains producing antibiotics related to 2 as
discussed in the following communication.
material representing aromatization unaccompanied by
hydrolysis was isolated—an observation consistent with the
known rapid hydrolysis of N-acylpyrroles.[10] While 2 is
sensitive to acid, base, and elevated temperatures, it survives
for days at room temperature in aqueous solution under
neutral pH conditions. Two structural features of 2 undoubt-
ꢀ
edly contribute to its modest persistence: the C3 H bond is
shown in energy-minimized structures of 2 not to be aligned
with the p bond of the C1’ carbonyl group, and this lack of
conjugation creates a kinetic barrier to proton removal (see
structure 9). The out of conjugation conformation is at least
partially enforced by hydrogen bonds from asparagine-
derived NH groups to the C5 carbonyl oxygen atom. The
electron-withdrawing effect of the C5 carbonyl group also
diminishes the thermodynamic benefit of aromatization, and
this electron-withdrawing effect is enhanced by the same
hydrogen bonds.
With the structure of 2 established, we addressed how
such a polar molecule gains entry into E. amylovora cells and
the nature of its molecular target.[11] Addition of the synthetic
tripeptide Ala-Gly-Gly to the test medium, in amounts
exceeding three times that of 2, suppresses the antibiotic
activity of 2. Thus, 2 likely gains entry through the tripeptide
transporter, and the added tripeptide monopolizes the trans-
porter thereby limiting cellular entry of 2.[6,11]
Experimental Section
Cultivation and isolation: E. coli XL2Blue-pUC449 was grown in a
shaker maintained at 308C for 24 h in minimal media supplemented
with 100 mgmLꢀ1 ampicillin.[5] The culture supernatant was applied to
a Dowex 1X8-200 (HCO3ꢀ) anion-exchange column and eluted with
CO2-saturated H2O. The active fractions were lyophilized, then
applied to a C18 solid-phase extraction column and eluted with H2O/
CH3CN (100/0 to 90/10). Further fractionation by HPLC, first with a
reverse-phase column (Vydac, 218TP510) using H2O/0.1m NH4OAc/
CH3CN (94/5/1), then with a polyhydroxyethyl A column (Poly LC,
Inc.) using H2O/0.1m NH4OAc/CH3CN (20/5/75) gave compounds 2
and 8 in yields of approximately 1 mgLꢀ1 and 0.2 mgLꢀ1, respectively.
Cloning of E. coli-pUC449: Pantocin A producing cosmid clone
pCPP702 was subcloned to generate pCPP1051, from which a XbaI-
HindIII fragment was further subcloned into pBluescript to yield
plasmid pCPP717.[5] The cloned insert was sequenced (GenBank
accession No. U81376). A 3.5 kb DNA fragment was then amplified
by a polymerase chain reaction from pCPP717 using primer (5’-
Since addition of histidine suppresses the activity of 2, the
cellular target was sought on the histidine biosynthetic
pathway.[11] The common strategy of feeding intermediates
CCGCATCTAGAGTAGGTATGAC-3’)
ATACTCTGCAGAGTTGGTGCTCCA-3’), and ligated into
pUC19 at the XbaI and PstI sites. Recombinants were selected
and
primer
(5’-
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Chemie
from transformed E. coli XL2 Blue cells on Luria–Bertani plates
supplemented with ampicillin at 100 mgmLꢀ1, to yield clone E. coli
XL2 Blue-pUC449.
(KBr): n˜max = 3700–2400 (br), 2955 (sh), 1671, 1528, 1447 cmꢀ1; ESI-
MS/MS: m/z: 313 [MþH]+, 181, 153, 125.
HPLC analysis was performed on an Aqua C18 column (250 ”
4.6 mm, 5 m, 125 Š, Phenomenex) with a mobile phase of H2O(0.1%
TFA)/CH3CN(0.1% TFA) with a gradient of 100/0 (0–5 min); 100/0
to 85/15 (5–20 min) at 1 mLminꢀ1. (3R,6R,9R)-3 and (3S,6S,9S)-3
showed base-line separation with retention times of 15.6 and
15.9 min, respectively. 3 derived from 2 co-migrated with (3S,6S,9S)-
3 but not (3R,6R,9R)-3.
2: [a]2D5 = ꢀ291.18 (c = 1.8, H2O); IR (KBr): n˜max = 3700–2400 (br),
1676, 1578, 1402 cmꢀ1; UV (H2O): lmax (e) = 200 (12089), 272 nm
(5447); HRFAB-MS: m/z: 331.1018 [MþNa]+, calcd m/z: 331.1018
for C13H16N4O5Na; ESI-MS/MS: m/z: 309 [MþH]+, 177, 159, 151, 149.
8: 1H NMR (500 MHz, D2O, 25 8C): d = 6.81 (1H,d, J = 3.7 Hz,
H3), 6.12 (1H, d, J = 3.7 Hz, H4), 4.66 (1H, dd, J = 9.0, 4.5 Hz,
H3’(Asn aH)), 3.75 (1H, t, J = 6.5 Hz, H3’’), 2.85 (1H, dd, J = 15.0,
4.5 Hz, H4’a), 2.80–2.69 (3H, m, H1’’ and H4’b), 2.25–2.10 ppm (2H,
m, H2’’); 13C NMR (100 MHz, 5%CD3OD in D2O, 25 8C): d = 178.6
(C6’, COOH), 177.0 (C5’, CONH2), 175.4 (C4’’) 163.4 (C1’), 137.4
(C5), 125.0 (C2), 113.5 (C3), 108.6 (C4), 55.3 (C3’’), 52.9 (C3’), 39.1
Received: January 29, 2003[Z5105]3
Keywords: antibiotics · gene expression · natural products ·
.
structure elucidation
(C4’), 31.5 (C2’’), 23.8 ppm (C1’’); ESI-MS/MS/MS: m/z:
3 27
[MþH]+, 310, 195, 177, 149, 132, 106; UV (H2O): lmax (e) = 200
(6438), 220 (2994, sh), 275 nm (10887); IR (thin film): n˜max = 3700–
[1] H. S. Aldwinckle, S. V. Beer, Hortic. Rev. 1979, 1, 423.
[2] J. M. Erskine, L. E. Lopatecki, Can. J. Microbiol. 1975, 21, 3 5.
[3] R. S. Wodzinski, J. P. Paulin, J. Appl. Bacteriol. 1994, 76, 603.
[4] C. A. Ishimaru, E. J. Klos, R. R. Brubaker, Phytopathology 1988,
78, 746.
2400 (br), 1668, 1604, 1533, 1404 cmꢀ1
.
Preparation of 3 from 2: A solution of 2 (2.4 mg) in deionized
H2O (300 mL) was treated with Pd/C (10 wt%, 0.66 mg) and stirred
under hydrogen at room temperature for 8 h. The reaction mixture
was filtered over celite, washed with deionized H2O (2 mL), and the
combined aqueous fractions were dried to give 1.7 mg of 3. 1H NMR
(500 MHz, D2O, 25 8C): d = 4.46 (1H, d, J = 9.0 Hz, H3), 4.40 (1H, dd,
J = 8.0, 5.0 Hz, H3’), 3.93 (1H, t, J = 8.5 Hz, H6), 3.74 (1H, m, H9),
2.76 (1H, dd, J = 15.0, 5.0 Hz, H4’a), 2.65 (1H, dd, J = 15.0, 8.0 Hz,
H4’b), 2.35 (1H, m, H7a), 2.25–2.10 (4H, m, H2a, 1a, 8a, 2b), 1.91
(1H, m, H7b), 1.67 (1H, m, H8b), 1.64 ppm (1H, m, H1b); 13C NMR
(100 MHz, D2O + 5%CD3OD, 258C): d = 177.8 (C6’), 176.6 (C5’),
173.6 (C1’), 169.5 (C5), 61.0 (C3), 59.2 (C9), 53.2 (C3’), 49.6 (C6), 38.6
(C4’), 32.3 (C1), 30.2 (C2), 26.2 (C8), 25.6 (C7); [a]2D5 = ꢀ57.88 (c =
[5] S. A. I. Wright, C. H. Zumoff, L. Schneider, S. V. Beer, Appl.
Environ. Microbiol. 2001, 67, 284.
[6] S. F. Brady, S. A. Wright, J. C. Lee, A. E. Sutton, C. H. Zumoff,
R. S. Wodzinski, S. V. Beer, J. Clardy, J. Am. Chem. Soc. 1999,
121, 11912.
[7] A. E. Sutton, J. Clardy, Org. Lett. 2000, 2, 319.
[8] A. E. Sutton, J. Clardy, J. Am. Chem. Soc. 2001, 123, 9935.
[9] H.-G. Lombart, W. D. Lubell, J. Org. Chem. 1996, 61, 9437.
[10] F. M. Menger, J. A. Donohue, J. Am. Chem. Soc. 1973, 95, 432.
[11] M. Jin, Ph.D. thesis, Cornell University (Ithaca), 2003.
[12] J. Cavanagh, W. J. Fairbrother, A. G. Palmer III, N. J. Skelton,
Protein NMR Spectroscopy: Principles and Practice, Academic
Press, New York, 1995.
0.16, H2O); UV (H2O): lmax (e) = 200 nm (2200); IR (thin film): n˜max
=
3700–2400 (br), 2954 (sh), 1668, 1529 (sh), 1448, 1392, 1342 cmꢀ1; ESI-
MS/MS: m/z: 313 [MþH]+, 181, 153, 125.
Chiral TLC: Chiral TLC analyses were performed on CHIRAL-
PLATE (Macherey-Nagel) using solvent A (CH3OH/H2O/CH3CN,
5/5/20) or B (acetone/H2O/CH3OH, 10/2/2). 3 (0.15 mg, 0.5 mmol) was
heated in 6m HCl (300 mL) at 858C for 19 h to give 4 and 5. Authentic
d- and l- Asn were also treated in the same manner. Compound 5
migrated the same distance as treated l-Asn (Rf = 0.47 in A, 0.66 in
B), which was easily distinguishable from treated d-Asn (Rf = 0.40 in
A, 0.58 in B)
3: As shown in Scheme 1, N-(PhF) l-or d-dimethyl glutamate
was used in a Claisen condensation, reductive amination, and lactam-
cyclization sequence to give enantiopure (3S,6S,9S)-7 or (3R,6R,9R)-7
following the procedure of Lombart and Lubell.[9] Coupling with tert-
butyl-l-asparagine ester followed by removal of protecting groups
then gave (3S,6S,9S)-3 or (3R,6R,9R)-3 in 7% overall yield.
(3S,6S,9S)-3: [a]2D5 = ꢀ50.78 (c = 1.01, H2O); H NMR (500 MHz,
1
D2O, 25 8C): d = 4.61 (1H,dd, J = 6.0, 7.0 Hz), 4.51 (1H, d, J = 8.8 Hz),
4.06 (1H, t, J = 8.5 Hz), 3.78 (1H, m), 2.87–2.77 (2H, m), 2.42 (1H,
m), 2.31–2.18 (3H, m), 2.13 (1H, m), 1.97 (1H, m), 1.73–1.63 ppm
(2H, m). (The small differences in d values between (3S,6S,9S)-3 and
3 derived from 2 are the result of differences in pH values and
concentration. Mixing two samples resulted in overlapping peaks).
13C NMR (100 MHz, D2O + 10%CD3OD, 258C): d = 175.9, 175.6,
174.0, 168.0, 60.8, 59.1, 51.1, 49.4, 37.5, 32.3, 30.2, 26.3, 25.0 ppm; IR
(thin film): n˜max = 3700–2400 (br), 2956 (sh), 1670, 1530 (sh), 1448,
1390, 1340 cm ꢀ1; ESI-MS/MS: m/z: 313 [MþH]+, 181, 153, 125.
(3R,6R,9R)-3: [a]D25 = + 45.78 (c = 0.98, H2O); 1H NMR
(500 MHz, D2O, 25 8C): d = 4.62 (1H, dd, J = 8.0, 5.0 Hz), 4.53(1H,
d, J = 8.8 Hz), 4.06 (1H, t, J = 8.5 Hz), 3.78 (1H, m), 2.84 (1H, dd, J =
15.5, 5.0 Hz), 2.71 (1H, dd, J = 15.5, 8.0 Hz), 2.42 (1H, m), 2.30–2.17
(3H, m), 2.05 (1H, m), 1.96 (1H, m), 1.75–1.64 ppm (2H, m);
13C NMR (100 MHz, D2O + 10% CD3OD, 258C): d = 175.9, 175.3,
173.9, 168.1, 60.8, 59.1, 50.9, 49.5, 37.6, 32.3, 30.6, 26.3, 25.0 ppm; IR
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