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Journal of Bacteriology, February 1999, p. 757-763, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate,
an Intermediate of the Phenanthrene-Degradative Pathway of
Nocardioides sp. Strain KP7
Kyoko
Adachi,1
Tokuro
Iwabuchi,2,*
Hiroshi
Sano,1,
and
Shigeaki
Harayama2
Marine Biotechnology Institute, Shimizu
Laboratories, Sodeshi, Shimizu, Shizuoka
424-0037,1 and
Marine Biotechnology
Institute, Kamaishi Laboratories, Heita, Kamaishi, Iwate
026-0001,2 Japan
Received 13 August 1998/Accepted 13 November 1998
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ABSTRACT |
1-Hydroxy-2-naphthoate (compound I) is a metabolite of the
phenanthrene-degradative pathway in Nocardioides sp. strain
KP7. This singly hydroxylated aromatic compound is cleaved by
1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the
ring cleavage product generated by the action of homogeneous
1-hydroxy-2-naphthoate dioxygenase was determined upon separation by
high-performance liquid chromatography at pH 2.5 by using nuclear
magnetic resonance (NMR) and mass spectroscopic techniques. The ring
cleavage product at this pH existed in equilibrium between two forms,
2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III)
and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the
structure of the major species became
(E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II;
common name, trans-2'-carboxybenzalpyruvate), which was in
equilibrium with compound III. Direct monitoring of the enzymatic
formation of the ring cleavage product by 1H-NMR in a
deuterated potassium phosphate buffer (pH 7.5) detected only compound
II as a product, and the proton on carbon 3 of compound II was not
exchanged with deuterium. Thus, compound II is likely to be the first
stable product of dioxygenation of 1-hydroxy-2-naphthoate.
| |
INTRODUCTION |
Biodegradation of polyaromatic
hydrocarbons (PAHs) attracts much attention because they are
contaminants frequently found in soils, aquifers, and sediments
(2). Phenanthrene is a PAH which has often been used as a
model for the biodegradation of PAHs (5). Two routes for
phenanthrene degradation by bacteria have been described (1, 4,
8-10, 13). In one route, phenanthrene is converted via
1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene, which is
ring-cleaved by 1,2-dihydroxynaphthalene dioxygenase and further
transformed to salicylate. In the other route, phenanthrene is
transformed to 1-hydroxy-2-naphthoate, which is ring cleaved by
1-hydroxy-2-naphthoate dioxygenase and further converted to o-phthalate.
The ring cleavage dioxygenases have an essential role in the
decomposition of singly and doubly hydroxylated aromatic compounds (6, 7). Several ring cleavage dioxygenases have been
purified and characterized, but few chemical structures of dioxygenase ring cleavage products have been characterized. The product of ring
cleavage of 1,2-dihydroxynaphthalene by 1,2-dihydroxynaphthalene dioxygenase has been determined by nuclear magnetic resonance (NMR)
analysis to be 2-hydroxychromene-2-carboxylate, which is further
transformed by 2-hydroxychromene-2-carboxylate isomerase to
trans-2'-hydroxybenzalpyruvate [IUPAC name,
(E)-4-(2-hydroxylatophenyl)-2-oxo-3-butenoate] (3). In phenanthrene degradation, the aromatic ring of
1-hydroxy-2-naphthoate is cleaved by 1-hydroxy-2-naphthoate dioxygenase
(10). The product of ring cleavage of 1-hydroxy-2-naphthoate
by 1-hydroxy-2-naphthoate dioxygenase exhibits a high level of
absorption at 300 nm, which is consistent with the presence of double
bonds in a substituent conjugated with an aromatic ring (1,
14). Since pyruvate and 2-carboxybenzaldehyde have been detected
as pathway intermediates, the chemical structure of the product of ring
cleavage of 1-hydroxy-2-naphthoate by 1-hydroxy-2-naphthoate
dioxygenase was suggested to be 2'-carboxybenzalpyruvate [IUPAC name,
4-(2-carboxylatophenyl)-2-oxo-3-butenoate] (1, 8, 9, 14)
(Fig. 1). However, the geometrical
structure of the ring cleavage product has not been investigated.
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FIG. 1.
Ring cleavage reaction of 1-hydroxy-2-naphthoate
catalyzed by 1-hydroxy-2-naphthoate dioxygenase. The ring cleavage
product is expected to be (Z)- or
(E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (1, 8, 9,
14).
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In the present study, we enzymatically produced the ring cleavage
product of 1-hydroxy-2-naphthoate by using 1-hydroxy-2-naphthoate dioxygenase, and the geometrical structure of the ring cleavage product
of 1-hydroxy-2-naphthoate was determined by NMR and fast atom
bombardment-mass spectrometry (FAB-MS) analyses.
| |
MATERIALS AND METHODS |
Chemical.
1-Hydroxy-2-naphthoic acid was purchased from
Tokyo Organic Chemicals (Tokyo, Japan).
Purification of 1-hydroxy-2-naphthoate dioxygenase.
The
pMKT290 plasmid is a derivative of pUC18 carrying the structural gene
for 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp.
strain KP7 (10, 11). Cells of Escherichia coli
JM109 containing pMKT290 were cultivated in 10 liters of L broth
containing 50 µg of ampicillin ml
1 (10).
Cells from the overnight culture were pelleted by centrifugation at
10,000 × g for 20 min at 4°C, resuspended in 65 ml
of a 10 mM Tris-H2SO4 buffer (pH 7.5), and
disrupted by passage through a precooled French pressure cell (Ohtake
Works, Tokyo, Japan) at a pressure of 800 kg/cm2. The cell
extract was centrifuged at 27,700 × g for 30 min at 4°C, and the supernatant was recentrifuged at 250,000 × g for 60 min at 4°C. The supernatant was filtered through a
Sterivex-HV filter (0.45-µm pore size; Millipore, Bedford, Mass.),
and loaded into an anion-exchange column (TSKgel DEAE-5PW, 21.5 mm
[inside diameter {i.d.}] by 150 mm; Tosoh, Tokyo, Japan) fitted
to a high-performance liquid chromatography (HPLC) system (Tosoh). The
protein was eluted by a linear gradient of 0 to 0.5 M
Na2SO4 in 300 ml of a 10 mM Tris-H2SO4 buffer (pH 7.5) at a flow rate of 5 ml min1. The eluate was collected in 5-ml fractions on
ice. Ammonium sulfate was added to the pooled fractions containing the
1-hydroxy-2-naphthoate dioxygenase activity to achieve a final
concentration of 0.6 M at 4°C, and proteins which precipitated were
removed by centrifugation at 27,700 × g for 30 min at
4°C. The 1-hydroxy-2-naphthoate dioxygenase activity was recovered in
the supernatant. The supernatant was filtered through a Millex-GV
filter (0.45-µm pore size; Millipore) and loaded into a
hydrophobic-interaction column (TSKgel Phenyl-5PW, 21.5 mm [i.d.] by
150 mm; Tosoh) pre-equilibrated with a 10 mM Tris-H2SO4 buffer (pH 7.5) containing 0.6 M
ammonium sulfate. Proteins were eluted from the column by a linear
gradient from 0.6 to 0 M ammonium sulfate in 60 ml of a 10 mM
Tris-H2SO4 buffer (pH 7.5) at a flow rate of 1 ml min1. Ammonium sulfate was added to the pooled
fractions containing the 1-hydroxy-2-naphthoate dioxygenase activity to
achieve a final concentration of 2 M at 4°C, and precipitated
proteins were dissolved in 20 ml of a 10 mM
Tris-H2SO4 buffer (pH 8.0) at 4°C. The
dissolved proteins were loaded on an anion-exchange column (Mono Q
HR5/5, 5 mm [i.d.] by 50 mm; Pharmacia, Uppsala, Sweden) fitted to an HPLC system (Tosoh). Finally, purified protein was eluted by a linear
gradient of 0 to 0.5 M Na2SO4 in 60 ml of a 10 mM Tris-H2SO4 buffer (pH 8.0) at a flow rate of
1 ml min1. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was carried out in a premade polyacrylamide
slab gel (Multigel Gel 4/20; Dai-ichi Pure Chemicals, Tokyo, Japan), as
described previously (10).
Purification and identification of the ring cleavage
product.
Purified recombinant 1-hydroxy-2-naphthoate dioxygenase
(2.7 mg) was added to 1,600 ml of a 10 mM Tris-HCl buffer (pH 7.5) containing 150 µM 1-hydroxy-2-naphthoate. The progression and completion of the reaction at room temperature were monitored spectrophotometrically by measuring A300
(1, 10, 14). After acidification of the reaction mixture
with approximately 2 ml of 10 N HCl to pH 2.5, the reaction mixture was
extracted with 800 ml of chloroform and then with 800 ml of ethyl
acetate. The chloroform and ethyl acetate phases were combined, and
organic solvents were removed in vacuo. The dried sample was dissolved in 10 ml of 10% aqueous methanol containing 0.1% (vol/vol)
H3PO4 and injected onto a C18
column (CapcellPak-C18 type SG120, 4.6 mm [i.d.] by 250 mm; Shiseido,
Tokyo, Japan) fitted to an HPLC system (Tosoh) at 25°C. The elution
profile was monitored at 280 nm. The ring cleavage product was eluted
by a linear solvent gradient of 10 to 90% (vol/vol) methanol in 15 ml
of ultrapure water containing 0.1% (vol/vol)
H3PO4 at a flow rate of 1 ml
min1. The reaction product was eluted at 50% (vol/vol)
methanol. Methanol was removed from the purified fraction in vacuo, the
dried sample was dissolved in D2O, and its pH was adjusted
with DCl or NaOD.
UV spectroscopy of the purified reaction product.
UV spectra
of the purified reaction product at different pH values were measured
by using a UV spectrophotometer (UV2000; Shimadzu, Kyoto, Japan) at
25°C. The purified sample was dissolved in a 50 mM potassium
phosphate buffer (pH 7.5) or a 50 mM potassium phosphate buffer
acidified by 10 N HCl to pH 2.5.
NMR and MS.
All NMR spectra were acquired on a Unity 500 spectrometer (Varian, Palo Alto, Calif.) at 30°C. Two-dimensional
experiments such as H,H-correlated spectrometry (COSY), heteronuclear
single-quantum coherence spectrometry (HSQC), and heteronuclear
multibond correlation spectrometry (HMBC) were used for structure
determination of the product. FAB-MS was done with a JMS-SX102 mass
spectrometer (JEOL, Tokyo, Japan).
Monitoring of enzymatic reaction by 1H-NMR.
Transformation of 1-hydroxy-2-naphthoate in the reaction catalyzed by
1-hydroxy-2-naphthoate dioxygenase was monitored by 1H-NMR
for 180 min at 30°C. Each data set was obtained after 32 scans. The
reaction was carried out in 700 µl of 50 mM deuterated potassium
phosphate buffer (pH 7.5) containing 0.56 mM 1-hydroxy-2-naphthoate and
0.5 mg of 1-hydroxy-2-naphthoate dioxygenase.
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RESULTS AND DISCUSSION |
Purification of 1-hydroxy-2-naphthoate dioxygenase from E. coli.
An extract of E. coli JM109(pMKT290) contained
1-hydroxy-2-naphthoate dioxygenase activity at 12 µmol
min1 mg of protein1. This enzyme was
purified from extracts of E. coli JM109(pMKT290) as shown in
Table 1. The enzyme activity was eluted
from the first anion-exchange column (DEAE column) by
Na2SO4 at 0.11 M, and the activity was eluted
from the second anion-exchange column (Mono Q column) by
Na2SO4 at 0.2 M. The enzyme activity was
broadly eluted from a phenyl column. After the second anion-exchange
chromatography, the purified enzyme gave a single protein band at 45 kDa by SDS-PAGE (Fig. 2). The specific
activity of the purified enzyme was 1.6 mmol min1 mg of
protein1. The transformation of 1-hydroxy-2-naphthoate by
the purified enzyme was spectrophotometrically examined, and the
spectrum of 1-hydroxy-2-naphthoate changed immediately to one showing a
maximal absorption at 300 nm (data not shown). Thus, it was concluded that the purified enzyme exhibits 1-hydroxy-2-naphthoate dioxygenase activity (1, 10, 14).
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FIG. 2.
SDS-PAGE of purified 1-hydroxy-2-naphthoate dioxygenase
(45 kDa) from extracts of E. coli JM109(pMKT290). Lanes: 1, molecular mass markers; 2, pooled active fractions after the second
anion-exchange column chromatography step (Mono Q column).
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Chemical structures of the ring cleavage products of
1-hydroxy-2-naphthoate.
The enzymatically prepared ring cleavage
product of 1-hydroxy-2-naphthoate was purified by reverse-phase HPLC.
The yield of the purified ring cleavage product was 70%, and the
purity of the purified product was 94.4% by HPLC analysis. UV spectra
of the purified product at pH 2.5 and 7.5 are shown in Fig.
3A and B, respectively. The UV spectrum
at pH 7.5 showed a high level of absorption at 300 nm; however, the
spectrum at pH 2.5 showed two absorptions at 274 and 281 nm (Fig. 3A
and B). These results indicate that the reaction product has different
forms at two pH values.
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FIG. 3.
UV spectra of the purified ring cleavage product at pH
2.5 (in a 50 mM potassium phosphate buffer acidified by 10 N HCl) (A)
and pH 7.5 (in a 50 mM potassium phosphate buffer) (B).
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The
1H- and
13C-NMR spectra of the product were
recorded as described in Materials and Methods. The assignment of
1H- and
13C-NMR signals of the ring cleavage
product at two different pH
values is shown in Table
2. The initial pH of the purified
material
was 2.5. At this pH, two molecular species,
2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate
(compound III
in Table
2 and Fig.
4) and
2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate
(compound IV in Table
2 and Fig.
4), were resolved by
1H-NMR,
13C-NMR, and other two-dimensional NMR
analyses including COSY,
HSQC, and HMBC. These structures were
supported by the negative
FAB-mass spectrum at pH 2.5 showing molecular
ions at
m/z values
of 219 and 237 (M-H)
. When
the solution was neutralized to pH 7.5 with NaOD, compound
IV was not
detected and compound III became a minor species. Instead,
(
E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II
in
Table
2, Fig.
1, and Fig.
4) was revealed as a major species.
The
(
E) form of compound II was deduced from the coupling
constant
of (
3J3-4) 16.4 Hz (Table
2). The negative FAB-mass spectrum of this
compound showed a molecular
ion at an
m/z of 219 (M-H)
. Thus, the ring
cleavage product of 1-hydroxy-2-naphthoate took
at least three
different structures at different pH values. Two
forms, compound III
and compound IV, were in equilibrium under
acidic conditions, while at
neutral pH, compound III and compound
II were in equilibrium, the
latter compound being the major species
(Fig.
5B).
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FIG. 4.
Structures of the ring cleavage products identified by
NMR and FAB-MS analyses. In a solution at pH 2.5, compounds III and IV
were revealed. When the pH of the solution was raised to pH 7.5, compounds II and III were revealed, with compound II being the major
species. Compound II,
(E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (common name,
trans-2'-carboxybenzalpyruvate); compound III,
2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate; compound IV,
2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate.
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FIG. 5.
1H-NMR signals of the freshly prepared ring
cleavage product of 1-hydroxy-2-naphthoate (pH 7.5) (A), the ring
cleavage product neutralized for 1 h at pH 7.5 after its
purification at pH 2.5 (B), and the same sample after incubation in
deuterated buffer for two more weeks at pH 7.5 (C).
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Direct monitoring of the formation of the reaction product by
1H-NMR.
The transformation of 1-hydroxy-2-naphthoate
to its ring cleavage product in a reaction catalyzed by
1-hydroxy-2-naphthoate dioxygenase was monitored in real time by
1H-NMR. The intensities of the signals corresponding to
1-hydroxy-2-naphthoate decreased as the reaction progressed. As shown
in Fig. 6, a single product,
(E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II),
was formed and was stable for at least 180 min under the experimental
conditions. In this reaction, 95% of the substrate (1-hydroxy-2-naphthoate; compound I) was converted to the reaction product (compound II). Thus, compound II is either the primary product
of the reaction catalyzed by 1-hydroxy-2-naphthoate dioxygenase or a
stable derivative of a hypothetical primary product.
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FIG. 6.
Time course of the 1-hydroxy-2-naphthoate dioxygenase
reaction monitored by 1H-NMR at pH 7.5 in 50 mM deuterated
potassium phosphate buffer.
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Deuterium exchange of the ring cleavage product.
The
1H-NMR spectrum of compound II (prepared as described
above) is presented in Fig. 5A. In Fig. 5B, the 1H-NMR
spectrum of the mixture of compound II and compound III obtained after
purification of the ring cleavage product at pH 2.5 and neutralization
to pH 7.5 is presented. The H4 signals in compound II (
, 8.05 ppm)
are different between Fig. 5A and B. In Fig. 5A, H4 is a doublet, and
in Fig. 5B a broad single peak has appeared inside the double peak for
H4. This result indicates that the proton at position 3 of compound II
was partly displaced by deuterium during the
acidification-neutralization cycle (Fig. 5B). The sample whose spectrum
is shown in Fig. 5B was kept in deuterated buffer at pH 7.5 for two
more weeks at room temperature in the dark, and a 1H-NMR
spectrum was again recorded (Fig. 5C). The spectrum in Fig. 5C
indicates that the proton at position 3 was exchangeable and was
completely displaced by deuterium after 2 weeks.
From the structure of 1-hydroxy-2-naphthoate (compound I), it is
reasonable to assume that the primary product is
(
Z)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate
(Fig.
1),
which is subsequently transformed into its (
E) isomer
(compound II) by a spontaneous or enzyme-catalyzed reaction. A
model
for (
Z)-(
E) nonenzymatic isomerization reactions
in D
2O
is depicted in Fig.
7.
If the reaction in Fig.
7 occurs, the proton
at position 3 of compound
II should be exchanged. In fact, the
transformation of compound III to
compound II in the deuterated
buffer displaced the proton at position 3 of the product (Fig.
5B). However, the proton at position 3 of compound
II formed by
the enzymatic reaction in the deuterated buffer was not
displaced
by deuterium (Fig.
5A and
6). From these observations, we
eliminated
the spontaneous nonenzymatic reactions following the
enzymatic
reaction with dioxygenase as shown in Fig.
7 from the
possible
(
Z)-(
E) isomerization mechanisms of the
ring cleavage product.
Probably, the (
Z)-(
E)
isomerization reaction is a part of the
catalytic mechanism of
1-hydroxy-2-naphthoate dioxygenase.
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FIG. 7.
Possible mechanisms of spontaneous isomerization of a
hypothetical primary product
[(Z)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate] to
(E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II)
in D2O. Compounds III and V are hypothetical intermediates
in the nonenzymatic conversion from
(Z)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate to
(E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate.
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The chemical structures of the three different ring cleavage products
have been determined. In the study of Eaton and Chapman
(
3),
the product was isolated without changing the pH value,
while in the
study of Junker et al. (
12), the product was isolated
at pH
1.5. It is advisable to isolate such products without acidification,
as
this treatment may provoke the isomerization demonstrated in
this
study.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge S. Miyachi for encouragement, Y. Itazawa for technical assistance, and F. Nishida for mass spectrometry operation.
This study was supported by grants from New Energy and Industrial
Technology Development Organization (Japan).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Shiseido
Research Center, 1050 Nippa, Kohoku-ku, Yokohama, Kanagawa 223-8553, Japan. Phone: 81-45-542-1337. Fax: 81-45-545-3434. E-mail:
toku.iwabuchi{at}nifty.ne.jp.
Present address: Kyowa Hakko Kogyo, 1-6-1 Ohtemachi, Chiyoda-ku,
Tokyo 100-8185, Japan.
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Journal of Bacteriology, February 1999, p. 757-763, Vol. 181, No. 3
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
