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Previous Article | Next Article 
J Bacteriol, February 1998, p. 945-949, Vol. 180, No. 4
Marine Biotechnology Institute, Kamaishi
Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026, Japan
Received 11 August 1997/Accepted 6 December 1997
trans-2'-Carboxybenzalpyruvate hydratase-aldolase was
purified from a phenanthrene-degrading bacterium,
Nocardioides sp. strain KP7, and characterized. The
purified enzyme was found to have molecular masses of 38 kDa by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel
filtration chromatography. Thus, the homotrimer of the 38-kDa subunit
constituted an active enzyme. The Km and
kcat values of this enzyme for
trans-2'-carboxybenzalpyruvate were 50 µM and 13 s Phenanthrene is a polyaromatic
hydrocarbon and has been used as a model for the biodegradation of
polyaromatic hydrocarbons (4, 10, 11). In bacteria,
phenanthrene is degraded via 1-hydroxy-2-naphthoate (2, 8,
18-20) (Fig. 1). The enzymatic steps involved in the transformation of phenanthrene to
1-hydroxy-2-naphthoate have been characterized (21, 29, 30).
They are very similar to those involved in the transformation of
naphthalene to salicylate (5-7, 11, 12, 23, 25, 31). In
many bacteria, one ring of 1-hydroxy-2-naphthoate is cleaved by
1-hydroxy-2-naphthoate dioxygenase (2, 16, 18, 20, 24) (Fig.
1), yielding trans-2'-carboxybenzalpyruvate (1).
Although the formation of 2-carboxybenzaldehyde and pyruvate during the
degradation of phenanthrene has been demonstrated in an
Aeromonas strain and gram-negative bacterial strain B156
(2, 18), enzymes involved in the transformation of
trans-2'-carboxybenzalpyruvate to 2-carboxybenzaldehyde have
not yet been characterized. In the 2-naphthoate catabolic pathway in
Burkholderia strain JT 1500, the initial substrate was shown
to be transformed to 2-carboxybenzaldehyde via
1-carboxy-2-naphthoate, and two enzymatic steps were proposed for the
conversion of the ring cleavage product of 1-hydroxy-2-naphthoate to
2-carboxybenzaldehyde (24). In the present study, we
purified and characterized the enzyme involved in the transformation of
trans-2'-carboxybenzalpyruvate from a phenanthrene-degrading
organism, Nocardioides sp. strain KP7 (17).
Furthermore, the structural gene for this enzyme was cloned and
sequenced.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Biochemical and Genetic Characterization of
trans-2'-Carboxybenzalpyruvate Hydratase-Aldolase from a
Phenanthrene-Degrading Nocardioides Strain
and
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
1, respectively.
trans-2'-Carboxybenzalpyruvate was transformed to
2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The
structural gene for this enzyme was cloned and sequenced; the length of
this gene was 996 bp. The deduced amino acid sequence of this enzyme
exhibited homology to those of
trans-2'-hydroxybenzalpyruvate hydratase-aldolases from
Pseudomonas putida PpG7 and Pseudomonas sp.
strain C18.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
View larger version (13K):
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FIG. 1.
Proposed routes for the degradation of phenanthrene in
Nocardioides sp. strain KP7. Compound names: 1, phenanthrene; 2, 1-hydroxy-2-naphthoate; 3, trans-2'-carboxybenzalpyruvate; 4, 2-carboxybenzaldehyde; 5, o-phthalate. 1-Hydroxy-2-naphthoate dioxygenase and its
structural gene were characterized previously (16). The ring
cleavage product of 1-hydroxy-2-naphthoate was determined to be
trans-2'-carboxybenzalpyruvate (1).
2-Carboxybenzaldehyde dehydrogenase and its structural gene were
characterized previously (15). In this study,
trans-2'-carboxybenzalpyruvate hydratase-aldolase and its
structural gene were characterized.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Bacterial strain and culture conditions. The phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, used in this study was previously described (17). This strain was cultivated in marine broth (Difco Laboratories, Detroit, Mich.) in the presence or absence of 0.1% (wt/vol) phenanthrene at 30°C.
Preparation of trans-2'-carboxybenzalpyruvate.
trans-2'-Carboxybenzalpyruvate was prepared enzymatically in
50 mM potassium phosphate (KP) buffer (pH 7.5) containing 45 µM
1-hydroxy-2-naphthoate and 5 µg of 1-hydroxy-2-naphthoate dioxygenase ml1; the latter was purified from Escherichia
coli containing recombinant plasmid pMKT290 by a previously
described method (15).
Enzyme assay.
As described in the Results section,
trans-2'-carboxybenzalpyruvate was converted to
2-carboxybenzaldehyde and pyruvate by the enzyme purified in this
study. We call this enzyme trans-2'-carboxybenzalpyruvate hydratase-aldolase. Under standard conditions, the activity of trans-2'-carboxybenzalpyruvate hydratase-aldolase was
spectrophotometrically measured at 300 nm in 50 mM KP buffer (pH 7.5)
containing 45 µM trans-2'-carboxybenzalpyruvate. The
difference between the extinction coefficient of
trans-2'-carboxybenzalpyruvate and that of the reaction
products (an equimolar mixture of 2-carboxybenzaldehyde and pyruvate)
at 300 nm was determined to be 17.3 mM1
cm1. The Michaelis-Menten kinetic parameters of
trans-2'-carboxybenzalpyruvate hydratase-aldolase for
trans-2'-carboxybenzalpyruvate were determined by measuring
the initial velocity of transformation under standard conditions,
except that various concentrations of the substrate were used. The
reverse reaction was spectrophotometrically measured in 50 mM KP buffer
(pH 7.5) containing 100 µM 2-carboxybenzaldehyde and 100 µM
pyruvate at 25°C.
Analysis of the products formed from trans-2'-carboxybenzalpyruvate. Five micrograms of purified trans-2'-carboxybenzalpyruvate hydratase-aldolase was added to 1 ml of KP buffer (pH 7.5) containing 52.1 nmol of trans-2'-carboxybenzalpyruvate, and the mixture was incubated at 25°C for 5 min. Aliquots (400 µl each) of this reaction mixture were used to determine the amounts of 2-carboxybenzaldehyde and pyruvate produced by applying 2-carboxybenzaldehyde dehydrogenase purified from Nocardioides sp. strain KP7 (15) and L-lactate dehydrogenase (Boehringer GmbH, Mannheim, Germany) (26), respectively. Both enzymatic reactions were carried out at 25°C, and the formation of NADH from NAD+ was spectrophotometrically measured.
2-Carboxybenzaldehyde produced from trans-2'-carboxybenzalpyruvate by the action of purified trans-2'-carboxybenzalpyruvate hydratase-aldolase was also analyzed with an octyldecyl silane column (type SG120 CapcellPak-C18; 4.6 by 250 mm; Shiseido, Tokyo, Japan) fitted to a high-performance liquid chromatography (HPLC) system (Tosoh, Tokyo, Japan). The product was analyzed at 25°C with a linear gradient of 50 to 90% (vol/vol) methanol in 30 ml of ultrapure water containing 0.1% (wt/vol) H3PO4 at a flow rate of 1 ml min1. In this experiment, 2-carboxybenzaldehyde
(Tokyo Chemical, Tokyo, Japan) was used as a standard.
Purification of trans-2'-carboxybenzalpyruvate
hydratase-aldolase.
trans-2'-Carboxybenzalpyruvate
hydratase-aldolase was purified from cells of strain KP7 that had been
grown for 48 h at 30°C in 20 liters of marine broth (Difco)
containing 0.1% (wt/vol) phenanthrene. The cells were pelleted by
centrifugation at 10,000 × g for 20 min at 4°C,
resuspended in 200 ml of 10 mM Tris-H2SO4 buffer (pH 7.5), and disrupted by two passages through a precooled French pressure cell (Ohtake Works, Tokyo, Japan) at a pressure of 76 MPa. The resulting cell extract was centrifuged at 27,700 × g for 30 min at 4°C, and the supernatant fluid was
recentrifuged at 250,000 × g for 60 min at 4°C. The
resulting supernatant was passed 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 by 150 mm; Tosoh) fitted
to an HPLC system (Tosoh). The protein was eluted with a linear
gradient of 0 to 0.5 M Na2SO4 in 150 ml of 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. trans-2'-Carboxybenzalpyruvate hydratase-aldolase was
eluted at a salt concentration of 0.26 M. Pooled fractions containing
the trans-2'-carboxybenzalpyruvate hydratase-aldolase
activity were dialyzed against 20 mM KP buffer (pH 7.5). The dialyzed
fractions were adjusted to a 0.6 M saturation of ammonium sulfate at
4°C, and the precipitated proteins were removed by centrifugation at
27,700 × g for 30 min at 4°C. The trans-2'-carboxybenzalpyruvate hydratase-aldolase activity
was recovered in the supernatant fluid. This supernatant was passed through a Millex-GV filter (0.45-µm pore size; Millipore) and loaded
into a hydrophobic interaction column (TSKgel Phenyl-5PW; 7.5 by 75 mm;
Tosoh) that had been preequilibrated with 20 mM KP buffer (pH 7.5)
containing 0.6 M ammonium sulfate. Proteins were eluted from the column
with a linear gradient of 0.6 to 0 M ammonium sulfate in 60 ml of 20 mM
KP buffer (pH 7.5) at a flow rate of 1 ml min1. Pooled
fractions containing the trans-2'-carboxybenzalpyruvate hydratase-aldolase activity were dialyzed against 10 mM
Tris-H2SO4 buffer (pH 8.0). The dialyzed sample
was passed through a Millex-GV filter (0.45-µm pore size; Millipore)
and loaded into an anion-exchange column (MonoQ HR5/5; 5 by 50 mm;
Pharmacia, Uppsala, Sweden) fitted to an HPLC system (Tosoh). The
protein was eluted with a linear gradient of 0 to 0.25 M
Na2SO4 in 60 ml of 10 mM
Tris-H2SO4 buffer (pH 8.0) at a flow rate of 1 ml min1. trans-2'-Carboxybenzalpyruvate
hydratase-aldolase was eluted at a salt concentration of 0.13 M.
1. The molecular mass of the enzyme was
determined from the mobility relative to that of protein molecular mass
standards (
-galactosidase, 465 kDa; immunoglobulin G, 150 kDa;
immunoglobulin G Fab fragment, 50 kDa; and myoglobin, 17 kDa)
(Boehringer). The protein concentration was determined by the method of
Bradford (3) by use of an assay kit (Bio-Rad Laboratories,
Richmond, Calif.) with bovine serum albumin as a standard.
Amino acid sequencing. The amino-terminal sequence of the purified enzyme was determined by Edman degradation with an automated protein sequencer (model 477; Perkin Elmer Applied Biosystems, Branchburg, N.J.).
Gene cloning, sequencing, and expression in E. coli. In a previous study, a 6.2-kb BamHI DNA fragment carrying the structural gene for 1-hydroxy-2-naphthoate dioxygenase (phdI) was cloned into pACYC184 to construct pMKT280 (15, 16). The downstream region of phdI was sequenced by use of a Taq DyeDeoxy terminator cycle sequencing kit and a model 373A DNA sequencer (Perkin Elmer Applied Biosystems).
A DNA fragment carrying the gene for trans-2'-carboxybenzalpyruvate hydratase-aldolase (phdJ) was amplified by use of Vent DNA polymerase (New England BioLabs, Beverly, Mass.) with a set of primers. One PCR primer, 5'-ACGATCAGGACGACATATGACGTCACC-3', was designed to add an NdeI site in the
16 to +11 region of
phdJ, while the other PCR primer,
5'-TGGCGGCAGGATCCTTCGATGATG-3', was designed to add a
BamHI site in the +1055 to +1078 region of phdJ. The amplified DNA fragment was digested with NdeI and
BamHI and cloned into pET-22b(+) (27) to
construct pMKT310. The DNA fragment cloned into pMKT310 was sequenced
to confirm the absence of any mutation in the amplified fragment.
E. coli BL21(DE3) containing pMKT310 was cultivated in 100 ml of L broth containing 50 µg of ampicillin ml1 until
the A660 reached 0.6. After the addition of 1 ml
of 100 mM isopropyl--D-thiogalactopyranoside (IPTG), the
cells were further cultivated for 2 h at 37°C. The resulting
cells were pelleted by centrifugation at 10,000 × g
for 20 min at 4°C, resuspended in 5 ml of 10 mM
Tris-H2SO4 buffer (pH 7.5), and disrupted by two passages through a precooled French pressure cell (Ohtake Works) at
a pressure of 76 MPa. The cell extract was centrifuged at 27,700 × g for 30 min at 4°C, and the resulting supernatant fluid was recentrifuged at 250,000 × g for 60 min at
4°C. The residual supernatant fluid was passed through a Sterivex-HV
filter (0.45-µm pore size; Millipore). Measurement of the
trans-2'-carboxybenzalpyruvate hydratase-aldolase activity
in the cell extract was carried out as described earlier.
Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL DNA databases as accession number D89988.
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Expression of trans-2'-carboxybenzalpyruvate
hydratase-aldolase in strain KP7.
The expression of
trans-2'-carboxybenzalpyruvate hydratase-aldolase was
examined in strain KP7 grown in marine broth in the presence and
absence of phenanthrene. The specific activities of
trans-2'-carboxybenzalpyruvate hydratase-aldolase were 0.008 µmol min1 mg of protein1 in the extract
of strain KP7 grown in the absence of phenanthrene and 0.08 µmol
min1 mg of protein1 in the presence of
phenanthrene. This result indicated that
trans-2'-carboxybenzalpyruvate hydratase-aldolase of strain
KP7 was induced by phenanthrene or its metabolite(s).
Purification and characterization of trans-2'-carboxybenzalpyruvate hydratase-aldolase. The purification procedure for trans-2'-carboxybenzalpyruvate hydratase-aldolase from strain KP7 grown on phenanthrene is summarized in Table 1. The enzyme activity was eluted from the first anion-exchange column at an Na2SO4 concentration of 0.26 M. In hydrophobic interaction chromatography, the enzyme activity was eluted at an ammonium sulfate concentration of 0.35 M. In the second anion-exchange column chromatography, the activity was eluted at an Na2SO4 concentration of 0.13 M. The purified sample after the second anion-exchange column chromatography gave a single protein band in SDS-PAGE. The molecular mass of trans-2'-carboxybenzalpyruvate hydratase-aldolase evaluated by gel filtration chromatography was 113 kDa (data not shown), and that determined by SDS-PAGE was 38 kDa (Fig. 2). Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme.
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1, respectively.
Determination of the trans-2'-carboxybenzalpyruvate hydratase-aldolase activity in different buffers at different pH values
indicated the optimum pH for this enzyme to be 7.5. The optimum
temperature for this enzyme was 45°C.
The products formed from trans-2'-carboxybenzalpyruvate by
the action of purified trans-2'-carboxybenzalpyruvate
hydratase-aldolase were quantitatively determined with reactions
catalyzed by 2-carboxybenzaldehyde dehydrogenase and
L-lactate dehydrogenase. It was found that 19.1 nmol of
2-carboxybenzaldehyde and 21.3 nmol of pyruvate were formed from 20.8 nmol of trans-2'-carboxybenzalpyruvate in the reaction catalyzed by trans-2'-carboxybenzalpyruvate
hydratase-aldolase. An HPLC analysis showed that one of the reaction
products had the same retention time as 2-carboxybenzaldehyde (data not
shown).
The reverse reaction of this enzyme was spectrophotometrically examined
with 1 ml of 50 mM KP buffer (pH 7.5) containing 100 nmol of
2-carboxybenzaldehyde, 100 nmol of pyruvate, and 1.5 µg of the
enzyme at 25°C. When the reaction had equilibrated, it was calculated
from the change in the absorbance at 300 nm that 21 nmol of
trans-2'-carboxybenzalpyruvate had been formed.
Assuming 1:1 stoichiometry for 2-carboxybenzaldehyde and pyruvate
consumption and the formation of
trans-2'-carboxybenzalpyruvate, the equilibrium constant of the reaction was calculated to be [(100 21)(100 21)]/21 = 297. When this value was used to
estimate the amounts of 2-carboxybenzaldehyde and pyruvate produced
from 20.8 nmol of trans-2'-carboxybenzalpyruvate at
equilibrium, the result was 19.5 nmol. This value was in agreement with
those determined experimentally (19.1 and 21.3 nmol, respectively).
The activity of this enzyme for trans-2'-hydroxycinnamate,
benzalpyruvate, and trans-2'-methoxybenzalpyruvate was
spectrophotometrically examined at a substrate concentration of 50 µM
in 50 mM KP buffer (pH 7.5) at 25°C. Under these conditions, this
enzyme transformed none of these compounds.
Effects of metals, chelators, and borohydride. Many bacterial aldolases require a divalent cation, such as Zn2+, for catalysis and are inhibited by EDTA (13, 28). Thus, the effects of metals and chelators on the activity of trans-2'-carboxybenzalpyruvate hydratase-aldolase were examined. EDTA at 10 mM, EGTA at 10 mM, Mn2+ at 1 mM, Mg2+ at 10 mM, and Ca2+ at 1 mM did not have any significant effect on the activity of trans-2'-carboxybenzalpyruvate hydratase-aldolase. Members of another type of aldolase, exemplified by the class I fructose-1,6-bisphosphate aldolase, form an intermediate with their substrate through a Schiff base, and borohydride irreversibly inhibits this type of aldolase by reducing the Schiff base (9). Preincubation of trans-2'-carboxybenzalpyruvate hydratase-aldolase in 50 mM KP buffer (pH 7.5) containing 20 mM sodium borohydride and 20 µM sodium pyruvate for 10 min at 25°C did not inhibit the activity of this enzyme. These observations indicated that the catalytic mechanism of this enzyme is different from those of metal-dependent and Schiff base-forming aldolases.
Amino-terminal sequence of trans-2'-carboxybenzalpyruvate hydratase-aldolase. The amino-terminal sequence of purified trans-2'-carboxybenzalpyruvate hydratase-aldolase was determined by automated Edman degradation to be Thr-Ser-Pro-Ala-Val-Thr-Ser-Ala-Asp-Ile-Thr-Gly-Leu-Val-Gly-Ile-Val-Pro-Thr-Pro-Ser-Lys-Pro-Gly.
Nucleotide sequence of the gene coding for trans-2'-carboxybenzalpyruvate hydratase-aldolase. We had previously cloned and sequenced the gene coding for 1-hydroxy-2-naphthoate dioxygenase (phdI) from strain KP7 (16). An open reading frame had been found downstream of phdI. The amino-terminal sequence deduced from this open reading frame was in perfect agreement with the amino-terminal sequence determined for purified trans-2'-carboxybenzalpyruvate hydratase-aldolase. We then sequenced the open reading frame using primers designed from the determined sequence. The length of the trans-2'-carboxybenzalpyruvate hydratase-aldolase gene, called phdJ, was 996 bp, and the deduced amino acid sequence for the enzyme was 332 amino acids long. The molecular mass (35 kDa) of the enzyme calculated from the deduced amino acid sequence was in agreement with the size determined by SDS-PAGE (38 kDa).
The deduced amino acid sequence of this enzyme exhibited significant similarities (40% identity) to those of NahE and DoxI (5-7) (Fig. 3). NahE is encoded by the nahE gene on naphthalene catabolic plasmid NAH7 in Pseudomonas putida PpG7, while DoxI is encoded by the doxI gene in dibenzothiophene-degrading strain Pseudomonas sp. strain C18. NahE has been found to exhibit trans-2'-hydroxybenzalpyruvate hydratase-aldolase activity (6, 7). The amino-terminal sequence of trans-2'-hydroxybenzalpyruvate hydratase-aldolase from naphthalenesulfonate-degrading strain P. vesicularis BN6 (22) is also similar to that of trans-2'-carboxybenzalpyruvate hydratase-aldolase. trans-2'-Hydroxybenzalpyruvate hydratase-aldolase from strain BN6 has a molecular mass of 120 kDa and is composed of identical subunits of 38.5 kDa. Clearly, trans-2'-hydroxybenzalpyruvate hydratase-aldolases in these strains and trans-2'-carboxybenzalpyruvate hydratase-aldolase in strain KP7 are evolutionarily related. On the other hand, no significant sequence similarity was observed between trans-2'-carboxybenzalpyruvate hydratase-aldolase and other aldolases. Thus, trans-2'-carboxybenzalpyruvate hydratase-aldolase and trans-2'-hydroxybenzalpyruvate hydratase-aldolase constitute a novel enzyme family, and it may be of interest to clarify the reaction mechanism of these enzymes in future studies.
|
Expression of the trans-2'-carboxybenzalpyruvate
hydratase-aldolase gene in E. coli.
A set of PCR
primers was designed to amplify a DNA fragment containing
phdJ, and a 1,094-bp DNA fragment was amplified by use of
Vent DNA polymerase and these primers. The amplified fragment was
cloned into pET-22b(+) to construct pMKT310. The nucleotide sequence of
the cloned fragment was identical to that of the original phdJ sequence, except for the primer regions. E. coli BL21(DE3) containing pMKT310 was cultivated in the presence
or absence of 0.1 mM IPTG in L broth at 37°C, and the
trans-2'-carboxybenzalpyruvate hydratase-aldolase activity
in the extracts of the cells thus cultivated was measured. The activity
of trans-2'-carboxybenzalpyruvate hydratase-aldolase was not
detected in the extract of noninduced L broth-grown cells (<0.001
µmol min1 mg of protein1), while it was
expressed at 0.04 µmol min1 mg of
protein1 in IPTG-induced cells. The results indicate that
the 996-bp phdJ gene encodes
trans-2'-carboxybenzalpyruvate hydratase-aldolase.
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ACKNOWLEDGMENTS |
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We thank R. W. Eaton for generously providing benzalpyruvate and trans-2'-methoxybenzalpyruvate. We are also grateful for the support and valuable advice of Shigetoh Miyachi and Jun Inoue and for the technical assistance of Yukiko Itazawa and Yoshio Sasaki.
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FOOTNOTES |
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* Corresponding author. Mailing address: Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026, Japan. Phone: 81-193-26-6544. Fax: 81-193-26-6592. E-mail: harayama{at}kamaishi.mbio.co.jp.
Present address: Shiseido Research Center, 1050 Nippa, Kohoku-ku,
Yokohama, Kanagawa 223, Japan.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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