From the Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026, Japan
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ABSTRACT |
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1-Hydroxy-2-naphthoate dioxygenase, which cleaves
the singly hydroxylated aromatic ring, was purified from
phenanthrene-degrading Nocardioides sp. strain KP7. The
purified enzyme had a molecular mass of 45 kDa by SDS-polyacrylamide
gel electrophoresis and 270 kDa by gel filtration chromatography. The
apparent Km and kcat values
of this enzyme for 1-hydroxy-2-naphthoate were 10 µM and
114 s1, respectively. One mole of molecular oxygen was
consumed when 1 mol of 1-hydroxy-2-naphthoate was oxidized. This enzyme
contained 1 mol of Fe(II)/mol of the subunit and was inactivated by
o-phenanthroline. The enzyme that had been inactivated by
o-phenanthroline was reactivated by incubating with
FeSO4 and ascorbic acid. Thus, Fe(II) was required for the
enzyme to exhibit activity. The structural gene for this enzyme was
screened from a cosmid library and then sequenced, the length of the
1-hydroxy-2-naphthoate gene being 1161 base pairs. The deduced amino
acid sequence of this enzyme was different from those of other
ring-cleaving dioxygenases that cleave the doubly hydroxylated aromatic
ring.
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INTRODUCTION |
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Dioxygenases are a group of enzymes that catalyze the incorporation of two atoms of molecular oxygen into a substrate (1). These enzymes play an important role in the aerobic decomposition of aromatic compounds in bacteria. The aromatic ring of the substrate is generally hydroxylated by either a ring-hydroxylating dioxygenase or a ring-hydroxylating monooxygenase, and the intermediate possessing two hydroxyl groups is subsequently cleaved by a ring-cleaving dioxygenase (2, 3).
Dioxygenases cleaving an aromatic ring that possesses two adjacent hydroxyl groups are divided into two classes. One class of enzymes, the extradiol type, cleaves the ring at a bond proximal to one of the two hydroxylated carbon atoms. The other class of enzymes, the intradiol type, cleaves the aromatic ring between two hydroxylated carbon atoms (1). In the case of gentisate containing two hydroxyl groups at a position para to each other, the ring is cleaved between carbon 1 and carbon 2 by gentisate 1,2-dioxygenase, and the ring-fission mechanism is believed to resemble that of extradiol-type fission (4, 5). Extradiol dioxygenases and gentisate 1,2-dioxygenase contain non-heme ferrous iron, whereas intradiol dioxygenases contain non-heme ferric iron (6). Non-heme iron is essential for the activity of these ring-cleaving dioxygenases.
In bacterial pathways for the degradation of phenanthrene via o-phthalate, the initial substrate is transformed to 1-hydroxy-2-naphthoate, which is further oxidized by the ring cleavage enzyme, 1-hydroxy-2-napthoate dioxygenase (Fig. 1) (7-10). The ring cleavage of singly hydroxylated 1-hydroxy-2-naphthoate is distinctive from other ring cleavage reactions in which the substrate always contains two hydroxyl groups. Despite its biochemically interesting properties, only a preliminary characterization of 1-hydroxynaphthoate dioxygenase has been presented (8, 10).
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In this study, we purified and characterized 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. strain KP7 and cloned and sequenced the structural gene for this enzyme.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strain-- Nocardioides sp. strain KP7, which degrades phenanthrene via o-phthalate (11), was used in this study.
Enzyme Assay--
The activity of 1-hydroxy-2-naphthoate
dioxygenase under standard conditions was spectrophotometrically
measured at 300 nm in 50 mM Tris-HCl (pH 7.5) containing
0.1 mM 1-hydroxy-2-naphthoate at 25 °C. The absorbance
of 1-hydroxy-2-naphthoate was a maximum at 340 nm, and the
transformation of this substrate to
trans-2'-carboxybenzalpyruvate resulted in the appearance of
another peak at 300 nm, as has been reported by Kiyohara and Nagao (8)
and Barnsley (10). The difference between the extinction coefficient of
trans-2'-carboxybenzalpyruvate (the ring cleavage product of
1-hydroxy-2-naphthoate) and that of 1-hydroxy-2-naphthoate at 300 nm
was determined to be 11.5 mM1
cm
1. The specific activity of this enzyme is expressed as
micromoles of trans-2'-carboxybenzalpyruvate formed per
min/mg of protein. The Michaelis-Menten kinetic parameters of this
enzyme for 1-hydroxy-2-naphthoate were obtained by measuring the
initial velocities that were determined under these conditions, except
that various concentrations of the substrate were used. The inhibition
of 1-hydroxy-2-naphthoate dioxygenase by substrate analogues was
analyzed similarly in the presence of the analogues at concentrations
of 1-1000 µM.
Purification of 1-Hydroxy-2-naphthoate
Dioxygenase--
1-Hydroxy-2-naphthoate dioxygenase was purified from
extracts of strain KP7. Cells were grown for 48 h at 30 °C in
10 liters of marine broth (Difco) containing 0.1% (w/v) phenanthrene.
Cell extracts were prepared as described previously (12) and loaded into an anion-exchange column (TSKgel DEAE-5PW, 21.5 × 150 mm; Tosoh, Tokyo, Japan) fitted to a high-performance liquid chromatography system (Tosoh). The protein was eluted by a linear gradient of 0-0.5
M Na2SO4 in 300 ml of a 20 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. 1-Hydroxy-2-naphthoate dioxygenase was eluted at a
salt concentration of 0.1 M. Pooled fractions containing
the 1-hydroxy-2-naphthoate dioxygenase activity were adjusted to 14.6%
saturation of ammonium sulfate at 4 °C, and the proteins that had
been 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 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, 21.5 × 150 mm; Tosoh) that had been pre-equilibrated
with a 20 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 the 20 mM
Tris-H2SO4 buffer (pH 7.5) at a flow rate of 1 ml min
1. Pooled fractions containing active
1-hydroxy-2-naphthoate dioxygenase were concentrated to 80 µl in a
1.5-ml centrifuge tube fitted with an Ultrafree C3-LGC membrane
(Millipore), loaded into a gel filtration column (TSKgel G3000SWXL,
7.8 × 300 mm; Tosoh), and eluted with a mobile phase of 20 mM Tris-H2SO4 (pH 6.8) containing 100 mM Na2SO4 at a flow rate of 0.5 ml min
1. The absorbance of the protein effluent was
monitored at 280 nm.
Measurement of Oxygen Uptake-- The oxygen uptake catalyzed by 1-hydroxy-2-naphthoate dioxygenase was measured at 25 °C by an oxygen electrode (type 5/6 Oxygraph; Gilson, Villiers-le-Biel, France). Five µl of 10 mM 1-hydroxy-2-naphthoate was added to a cuvette containing 1.5 ml of 50 mM Tris-HCl (pH 7.5), and 36 µl of the enzyme solution (49 pmol) was subsequently added to the cuvette. The oxygen consumption was then measured, the initial oxygen concentration in the buffer being assumed to be 250 µM.
Determination of Iron Content-- The iron content of 1-hydroxy-2-naphthoate dioxygenase was determined by using o-phenanthroline in the presence of 0.1 mM ascorbate (14).
Amino-terminal Sequencing-- The amino-terminal sequence of the purified enzyme was determined by Edman degradation with an automated protein sequencer (Perkin-Elmer model 477).
Gene Cloning and Sequencing-- Two degenerate PCR1 primers (the N primer and cC primer), which could collaboratively amplify a 75-bp-long DNA fragment encoding the amino-terminal 25 amino acid residues of 1-hydroxy-2-naphthoate dioxygenase, were designed. Their sequences were 5'-AC(TCAG)(TC)T(TCAG)GA(AG)GC(TCAG)GC (TCAG)CA(AG)GC(TCAG)GC-3' for the N primer, and 5'-CAT(TCAG)A(AG)(AG)TG(TC)TC(TC)TC(TCAG)GC(AGT)ATCCA-3' for the cC primer. Cloning of the amplified PCR product was carried out by using a pCR-ScriptTM SK(+) cloning kit (Stratagene Cloning Systems, La Jolla, CA), and the nucleotide sequence of the cloned PCR product was determined by using a Taq DyeDeoxy terminator cycle sequencing kit and a 373A DNA sequencer (Perkin-Elmer).
Enhanced chemiluminescence (ECL)-labeled probes were prepared by using the 75-bp-long PCR product as a template and ECL probe-amp reagents (Amersham Pharmacia Biotech). A cosmid library was prepared by using the BamHI-cleaved pLAFR3 vector and total DNA of strain KP7 that had been partially cleaved by Sau3AI according to the method of Staskawicz et al. (15). The colonies of the library were screened by hybridizing with the ECL-labeled probe. PCR amplification of the long fragments was carried out by using a GeneAmp XL PCR kit (Perkin-Elmer). Sequencing of the cloned gene for 1-hydroxy-2-naphthoate dioxygenase was carried out as described. ![]() |
RESULTS |
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Purification and Characterization of 1-Hydroxy-2-naphthoate
Dioxygenase--
1-Hydroxy-2-naphthoate dioxygenase was induced by
phenanthrene in strain KP7; the specific activity of the enzyme in an
extract of strain KP7 was 8.4 µmol min1 mg of
protein
1 after growth on marine broth containing 0.1%
(w/v) phenanthrene, whereas it was 1.0 µmol min
1 mg of
protein
1 after growth on marine broth without
phenanthrene. This enzyme was purified from extracts of strain KP7
grown on phenanthrene as summarized in Table
I. The enzyme activity was eluted from a
DEAE column at a Na2SO4 concentration of 0.14 M, and separation of the pooled DEAE fractions by a phenyl
column eluted the enzyme activity at an ammonium sulfate concentration
of 0.27 M. 1-Hydroxy-2-naphthoate dioxygenase was further
separated by gel filtration chromatography, the purified sample giving
a single protein band by SDS-polyacrylamide gel electrophoresis (Fig.
2). The molecular mass of
1-hydroxy-2-naphthoate dioxygenase evaluated by gel filtration
chromatography was 270 kDa (data not shown), and that by
SDS-polyacrylamide gel electrophoresis was 45 kDa (Fig. 2). Thus, the
homohexamer of the 45-kDa subunit constituted an active enzyme.
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Effects of Chelators and Metals on the Activity of 1-Hydroxy-2-naphthoate Dioxygenase-- The effect of various metals and metal chelators on the activity of the purified enzyme was examined (Table II). 1-Hydroxy-2-naphthoate dioxygenase was inactivated by 0.1 mM o-phenanthroline, which is the chelator specific to ferrous iron, but none of the other chelators and metals had any strong influence on the activity of this enzyme. Reactivation of the enzyme that had been inactivated by o-phenanthroline was examined with several metals in the presence of 0.1 mM ascorbate (Table III). Only Fe(II) was effective in reactivating the inactivated 1-hydroxy-2-naphthoate dioxygenase, which indicated that Fe(II) was a cofactor of this enzyme.
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Iron Content of Purified 1-Hydroxy-2-naphthoate Dioxygenase-- 1-Hydroxy-2-naphthoate dioxygenase was found to contain 1.15 mol of iron/mol of the subunit.
Amino-terminal Sequence of 1-Hydroxy-2-naphthoate Dioxygenase and Cloning and Nucleotide Sequence of Its Structural Gene-- The 53-residue amino-terminal sequence of purified 1-hydroxy-2-naphthoate dioxygenase was determined by automated Edman degradation to be Asn-Ser-Ser-Asn-Thr-Gly-Ala-Pro-Glu-Ala-Gln-Ala-Ala-Thr-Leu-Glu-Ala-Phe-Asp-Arg-Arg-Ala-Ala-Glu-Gln-Tyr-Leu-Arg-Gly-Gln-Trp-Ile-Ala-Glu-Glu-His-Leu-Met-Arg-Ala-Ile-Gly-Gly-Pro-Arg-Pro-Ala-Gly-Ile-Pro-Tyr-Trp-Glu. Based on this amino acid sequence, two degenerate PCR primers (the N primer and cC primer) that would allow amplification of the 75-bp-long product were designed as described under "Experimental Procedures." The primer set was used to amplify a PCR product corresponding to the size when total DNA from strain KP7 was used as a template. The 75-bp-long PCR product was cloned in the pCR-ScriptTM SK(+) plasmid, and the nucleotide sequence of this fragment was determined by cycle sequencing. The amino acid sequence deduced from the DNA sequence of this PCR product was in agreement with the amino-terminal sequence of 1-hydroxy-2-naphthoate. This result indicates that the amplified product corresponded to the partial sequence of the 1-hydroxy-2-naphthoate dioxygenase gene.
The pLAFR3-based cosmid library of strain KP7 was constructed, and colonies of the library were screened by the ECL-labeled PCR product that had been amplified from the 75-bp-long fragment. Restriction maps of the seven positive clones were constructed (Fig. 3). All positive clones were found to contain three BamHI fragments of 1.0, 1.8, and 6.2 kb in size. The N and cC primers amplified the 75-bp-long fragment only when the 6.2-kb BamHI fragment was used as a template. This result indicates that the 6.2-kb BamHI fragment encoded the amino-terminal region of 1-hydroxy-2-naphthoate dioxygenase. The 6.2-kb BamHI fragment was then subcloned into the BamHI site of pACYC184 to construct pMKT191.
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Expression of 1-Hydroxy-2-naphthoate Dioxygenase in Escherichia
coli--
The 2.8-kb StuI-BamHI-digested
fragment from pMKT191 (Fig. 3) was subcloned into pUC18 to construct
pMKT280. The SplI-BamHI fragment of pMKT280
downstream of the phdI gene was removed by digesting the
pMKT280 plasmid by SplI and BamHI and ligating
the digested DNA after the Klenow treatment. The pMKT290 plasmid thus constructed was used to transform E. coli JM109, and a cell
extract prepared from this transformant contained the
1-hydroxy-2-naphthoate dioxygenase activity of 20 µmol
min1 mg of protein
1. Thus it was confirmed
that the 1161-bp-long sequence encoded 1-hydroxy-2-naphthoate
dioxygenase.
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DISCUSSION |
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Substrates of ring-cleaving dioxygenases generally carry two hydroxyl groups at a position either ortho or para to each other (3). The biochemical reason for the requirement of two hydroxyl groups for ring cleavage is not yet understood. A model for the mechanism of extradiol dioxygenases has recently been proposed, and it was suggested that two hydroxyl groups of the substrate would interact with the catalytic iron, deprotonation of one of these hydroxyl groups being the first step of ring cleavage (16). 1-Hydroxy-2-naphthoate dioxygenase is unique among ring-cleaving dioxygenases because it can cleave a singly hydroxylated aromatic ring.
3-Hydroxy-2-naphthoate was a competitive inhibitor of 1-hydroxy-2-naphthoate dioxygenase, whereas 2-hydroxy-1-naphthoate and salicylate exhibited no effect on the enzyme activity. Comparing the structures of these compounds, a fused ring structure and the position of the carboxyl group seem to be important for binding to the substrate-binding pocket of this enzyme.
The previously characterized ring-cleaving dioxygenases required Fe(II) or Fe(III) for their activities. Fe(II) is required for extradiol-type ring-cleaving dioxygenases and gentisate 1,2-dioxygenase, whereas Fe(III) is required for intradiol-type ring-cleaving dioxygenases (1). In this study, we have demonstrated that 1-hydroxy-2-naphthoate dioxygenase also required Fe(II) for its activity, as has been suggested by Barnsley (10). However, the amino acid sequence of this enzyme was not related to that of any previously characterized ring-cleaving enzyme. 1-Hydroxy-2-naphthoate dioxygenase cleaved the aromatic ring between the carboxylated and hydroxylated carbons of the substrate. Such cleavage has also been observed in the reaction catalyzed by gentisate 1,2-dioxygenase. The subunit size of gentisate 1,2-dioxygenase from Comamonas acidovorans and C. testosteroni was approximately 40 kDa, which is close to that of 1-hydroxy-2-naphthoate dioxygenase (4, 5). Although 1-hydroxy-2-naphthoate dioxygenase was not able to cleave the ring of gentisate, it is still possible that the structure and catalytic mechanism of 1-hydroxy-2-naphthoate dioxygenase are similar to those of gentisate 1,2-dioxygenase. In this respect, it will be of interest to determine the primary structure of gentisate 1,2-dioxygenase.
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ACKNOWLEDGEMENTS |
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We are grateful to Shigetoh Miyachi for support. We also thank Atsushi Saitoh, Jun Inoue, and Satoshi Odo for valuable discussions and Yukiko Itazawa for technical assistance.
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FOOTNOTES |
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* This work was supported by the New Energy and Industrial Technology Development Organization.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000735.
Current address: Pharmaco Science Research Laboratories, Shiseido
Research Center, 1050 Nippa, Kohoku-ku, Yokohama, Kanagawa 223, Japan.
§ To whom correspondence should be addressed. Tel.: 81-193-26-6544; Fax: 81-193-26-6592; E-mail: sharayam{at}supernig.nig.ac.jp.
1 The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s).
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REFERENCES |
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