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Originally published In Press as doi:10.1074/jbc.M210240200 on April 2, 2003

J. Biol. Chem., Vol. 278, Issue 24, 21483-21492, June 13, 2003
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Characterization of a Novel Thermostable Mn(II)-dependent 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from a Polychlorinated Biphenyl- and Naphthalene-degrading Bacillus sp. JF8*

Takashi Hatta {ddagger} §, Gouri Mukerjee-Dhar ¶, Jiri Damborsky ||, Hohzoh Kiyohara {ddagger} and Kazuhide Kimbara ¶

From the {ddagger} Research Institute of Technology, Okayama University of Science, 401-1 Seki, Okayama 703-8232, Japan, Environmental Biotechnology Laboratory, Railway Technical Research Institute, Kokubunji, Tokyo 185-8540, Japan, || National Centre for Biomolecular Research Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

Received for publication, October 7, 2002 , and in revised form, March 14, 2003.
   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 ± 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 °C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 °C and 81 min at 75 °C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0–4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mM 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 µM for 2,3-dihydroxybiphenyl (at 60 °C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The catabolic versatility exhibited by microorganism plays an essential role in the carbon cycle, and this depends to a large extent on the use of oxygenases. In the degradation of aromatic compounds, oxygenases play a significant role both by hydroxylating the aromatic ring and by catalyzing the ring fission reaction. Nearly all bacterial pathways for the degradation of aromatic compounds transform initial substrates into intermediates that carry two or more hydroxyl groups on the aromatic ring, which are then substrates for the ring cleavage dioxygenases. Cleavage is generally catalyzed by metalloenzymes of one of the two functional classes: intradiol dioxygenases, which cleave ortho to the hydroxyl substituents, or extradiol dioxygenases, which cleave meta to the hydroxyl substituents.

Harayama and Rekik (1) proposed that extradiol dioxygenases could be divided into two families, those exhibiting a preference for bicyclic substrates and those with a preference for monocyclic substrates. Since then, several extradiol dioxygenases have been sequenced and characterized, and the evolutionary relationship among them has been investigated. The three-dimensional structures of three Type I extradiol dioxygenases, two of which cleave bicyclic compounds (2, 3) and one of which cleaves monocyclic compounds (4), have been reported. Whereas a majority of the bacterial extradiol dioxygenases that have been characterized contain Fe(II) as a catalytic metal center, there are only three known bacterial 3,4-dihydroxyphenylacetate 2,3-dioxygenase that utilize metals other than Fe(II); the enzyme from Bacillus brevis (5) and Arthrobacter globiformis CM-2 (6) are manganese-dependent, whereas that from Klebsiella pneumoniae exhibits magnesium dependence (7).

The degradation of biphenyl by bacteria has been well characterized at the genetic and biochemical level (8–10). The major pathway for biphenyl degradation is a four-step process initiated by the insertion of two atoms of oxygen at carbon positions 2 and 3 of the aromatic ring by biphenyl dioxygenase, the product of bphA genes. The resulting 2,3-dihydrodiol is dehydrogenated by a dihydrodiol dehydrogenase, the product of the bphB gene, to 2,3-dihydroxybiphenyl. This is cleaved at the meta position by the extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), the product of the bphC gene (Fig. 1). Then a hydrolase encoded by the bphD gene hydrolyzes the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid into benzoate and 2-hydroxypenta-2,4-dienoate.



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  		FIG. 1.
The meta-cleavage reaction catalyzed by the BphC enzyme.

 

This pathway has been well studied for its potential to cometabolize polychlorinated biphenyls (PCB),1 a family of recalcitrant, toxic environmental pollutants. Considerable differences have been found in congener selectivity pattern and range of activity among various PCB-degrading bacteria (11, 12). Although it is the initial oxygenase (BphA) that is crucially responsible for recognition and binding of the substrate (13), the ability of the bph pathway is also limited in parts by the BphC enzyme, which is incapable of transforming certain chlorinated dihydroxy biphenyls (14, 15) and is inhibited by 3-chlorocatechol (16–18). Extradiol dioxygenases are also susceptible to mechanism-based inactivation by their aromatic substrates. Therefore, the potential of this pathway for the remediation of PCB-contaminated soils may not be fully realized until more stable forms of the enzymes are available. Thermophilic bacteria produce enzyme variants with vastly improved stability. To be able to rationally engineer such properties into mesophilic enzymes, a study of the determinants of the stability is an important task for basic and applied research. Although thermophiles degrading aromatic compounds such as BTEX (benzene, toluene, ethylbenzene, xylene isomers) and phenol/cresol have been isolated (19, 20, 21), the aromatic pathways in these organisms are not well studied, and there have been few reports on the characterization of the genes/proteins involved (22, 23).

We have recently isolated a thermophilic bacterium Bacillus sp. JF8, which besides utilizing biphenyl and naphthalene as the sole source of carbon and energy, can transform several PCB congeners (24). From our analysis of the chlorobenzoic acids produced during mineralization of selected PCB congeners by strain JF8, we had concluded that the less chlorinated ring was oxidized, indicating similarity to the mesophilic PCB-degrading pathway. Probably, the upper biphenyl/PCB metabolic pathway in the thermophilic strain JF8 is identical to the metabolic pathway in mesophilic biphenyl/PCB degraders. Strain JF8N, a spontaneous mutant that lost the ability to utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, had indicated the presence of multiple dioxygenases in Bacillus sp. JF8. Here we report on the cloning and characterization of the extradiol dioxygenase, involved in the meta-cleavage of the biphenyl ring. To the best of our knowledge, this is the first report of a Mn(II)-dependent, thermostable 2,3-dihydroxybiphenyl 1,2-dioxygenase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Media, and Cloning of bphC
Bacillus sp. strain JF8 was grown on Castenholz D medium (24). Biphenyl was added at a concentration of 2 g/liter. For solid medium (1.5% agar), biphenyl was provided as vapor. Escherichia coli MV1190 was used as a host strain for DNA manipulation. Luria broth (pH 7.5) containing 10 g/liter tryptone, 5 g/liter yeast extract, and 5 g/liter NaCl was used as a complex medium. Ampicillin was added to the medium at a concentration of 100 µg/ml. Isopropyl-{beta}-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-{beta}-D-thiogalactopyranoside (X-gal) were used at concentrations of 100 and 40 µg/ml, respectively. Bacillus sp. JF8 was incubated at 60 °C, whereas Escherichia coli was incubated at 37 °C.

DNA Manipulation
Total DNA of strain JF8 was isolated by a modification of the procedure described by Marmur (25), and plasmid DNA was isolated by the alkaline lysis method (26). The total DNA of JF8 was digested by different restriction enzymes and DNA manipulations carried out as described by Sambrook et al. (27). The recovered DNA fragments were ligated into appropriate vectors and transformed into E. coli cells by the CaCl2 procedure (28). Transformants were selected on solid Luria broth plates supplemented with ampicillin, isopropyl-{beta}-D-thiogalactopyranoside, and X-gal by spraying with 2,3-dihydroxybiphenyl solution. Southern hybridization was performed under stringent conditions using Hybond N+ nylon membrane filter (Amersham Biosciences). DNA sequencing was carried out by the dideoxy chain termination method of Sanger et al. (29). The bphC gene of Rhodococcus sp. RHA1 (9) was introduced into the multicopy tac promoter vector, pTTQ18 (Amersham Biosciences), to give pTT122X. The MPC_mt2 expression plasmid, pIX121, was similarly constructed by inserting the xylE gene (4) encoded by the TOL plasmid pWWO into pTTQ18.

Purification and Characterization of the Native and Recombinant BphC Protein
The plasmid pQW1, constructed by inserting the bphC gene from strain JF8 into pTTQ18, was introduced into E. coli MV1190. Cells were grown in 1.5 liters of Luria broth containing 250 µg/ml of ampicillin to an A600 = 0.4. The bphC gene was induced by the addition of 1 mM isopropyl-{beta}-D-thiogalactopyranoside. After 4 h of induction, cells were collected by centrifuging at 4000 x g for 10 min. For the native BphC protein, cells of strain JF8 were grown overnight on Castenholz D medium or on Luria broth with biphenyl. The cells were washed twice with 25 mM phosphate buffer (pH 7.5) and resuspended in the same buffer. The cells were disrupted by a French pressure cell (Aminco Corp.) and centrifuged at 12,000 x g for 30 min and then at 105,000 x g for 60 min. The supernatant was used as a cell-free extract for assaying 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which was determined by measuring the formation of the meta-cleavage reaction product at 434 nm with a Beckman DU7500 spectrophotometer equipped with a thermocontrolled cuvette holder, and the pH of the Tris-buffer was set at the assay temperature. Enzymatic activity was assayed at 60 °C in 50 mM Tris-HCl buffer (pH 7.5) containing 330 µM 2,3-dihydroxybiphenyl. One unit of enzyme activity was defined as the amount of enzyme that converts 1 µmol of substrate/min. The molar extinction coefficient of the product under assay conditions was taken to be 13,200 cm–1 M–1. The relative meta-cleavage activities were determined from the extinction coefficients of the ring fission products formed from the following substrates: catechol ({lambda}max, 375 nm; e, 33,000 cm–1 M–1), 3-methylcatechol ({lambda}max, 388 nm; e, 32,000 cm–1 M–1), 4-methylcatechol ({lambda}max, 382 nm; e, 17,000 cm–1 M–1), and 4-chlorocatechol ({lambda}max, 379 nm; e, 40,000 cm–1 M–1). For purification of the enzyme, all manipulations were carried out at 10 °C in 25 mM potassium phosphate buffer (pH 7.5) containing 1 mM {beta}-mercaptoethanol (Buffer A) unless otherwise mentioned.

DEAE-Toyopearl Chromatography—The crude extract was loaded onto a DEAE-Toyopearl column (5.0 x 20 cm) previously equilibrated with Buffer A. Proteins were eluted with a linear gradient of KCl from 0 to 0.25 M in a total volume of 2500 ml of Buffer A. Active fractions, eluted around 0.2 M KCl, were collected.

Phenyl-Sepharose Column Chromatography—The collected fractions were dialyzed against Buffer A containing 1.2 M ammonium sulfate. The resulting protein solution was loaded onto phenyl-Sepharose HP 2.6/10 column (Amersham Biosciences) equilibrated with Buffer A containing 1.2 M ammonium sulfate. The enzyme was eluted with an 800-ml gradient of 1.2 to 0.0 M ammonium sulfate. The enzyme was eluted around 0.1 M ammonium sulfate.

MonoQ Column Chromatography—The active fractions eluted from phenyl-Sepharose column were pooled and dialyzed against Buffer A. The resulting solution was applied to a MonoQ HR 16/10 column (Amersham Biosciences) equilibrated with Buffer A. After the column was washed with 60 ml of Buffer A, the enzyme was eluted with 400 ml of a linear gradient from 0.0 to 0.5 M KCl. The enzyme was eluted around 0.3 M KCl.

Protein concentration was estimated by the method of Bradford (30) using bovine serum albumin as a standard. The purity and size of the enzyme proteins were estimated by SDS-PAGE according to the method of Laemmli (31). Protein staining of the gel was performed with Coomassie Brilliant Blue R-250.

Determination of Molecular Mass
The relative molecular mass of the native and recombinant enzyme was estimated by gel filtration on a Superdex 200HR 10/30 column (Amersham Biosciences) calibrated with ferritin (440 kDa), catalase (232 kDa), bovine serum albumin (67 kDa), and chymotrypsin (25 kDa). The relative subunit molecular mass was determined by SDS-PAGE. The Amersham Biosciences low molecular mass calibration kit was used.

N-terminal Sequence Analysis
Purified recombinant and native BphC_JF8 were subjected to N-terminal amino acid sequencing by the Edman degradation process using a model 477A protein sequencer (Applied Biosystems) in accordance with the manufacturer's procedure.

Kinetic Measurements
Michaelis-Menten kinetics of the reaction was verified by plotting reaction rates against substrate concentration. The Km and Vmax values were determined by nonlinear regression analysis of the plots and graphically from Lineweaver-Burk plotting of the initial cleavage rate. The substrate concentrations were in the range 0.05–0.25 µM 2,3-dihydroxybiphenyl at 60 °C and 0.1–0.5 µM at 25 °C. An activity assay of the enzyme was performed as mentioned above. For temperature stability and pH optimum, a temperature range of 30–90 °C and pH range of 6.0–9.0 (Tris-HCl and potassium phosphate buffer) was utilized, and the residual activities of the native and recombinant enzyme were determined. The buffer pH values were adjusted at the experimental temperature. The influence of metal cations, chelators, and inhibitors on enzyme activity was tested by incubating with samples of the purified enzyme (0.1 mg/ml) dialyzed against Tris-HCl buffer (pH 7.5) for different time intervals at 25 °C. The activation energy (Ea) was estimated using the Arrhenius equation for the temperature range of 30–75 °C. The value of Ea was determined from the slope of the straight line that resulted when the logarithm of the reaction constant, k, was plotted against 1/T.

Determination of Metal Content
Metal content was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a SEIKO SPQ6500 spectrometer (Seiko Instruments Inc.). Samples for ICP-MS were prepared using acid-washed glassware. Samples and standards were prepared in 0.1% HNO3. Separate standard curves were routinely prepared for iron and manganese, and samples were measured in quadruplicates.

EPR Sample Preparation and Spectroscopic Method
Samples of 0.3 ml were inserted in 4.0-mm inner diameter quartz tubes and frozen by slow immersion in liquid nitrogen. X-band EPR spectra were measured and analyzed using JEOL JES-CT470 (JEOL, Tokyo, Japan) equipped with a JEOL JES-G470 liquid helium variable temperature system. The EPR spectrometer setting was as follows: microwave power, 1 milliwatt; modulation amplitude, 1 mT at 100 kHz. Spectra were obtained with a microwave frequency of 8.93 GHz at 10 K as a single 4-min scan from 50 to 550 mT.

Phylogenetic Analysis
A homology search was performed with BLAST 2.0 (gapped BLAST) (32). Amino acid sequences retrieved from the protein databases were aligned using ClustalW version 1.9 (33). A phylogenetic tree was constructed by the neighbor-joining method using the Blosum62 distance matrix.

Molecular Modeling
Secondary structure predictions were done by building a consensus from the predicted results of PSIPRED (34), JPRED (35), PHD (36), and SSpro (37) methods. The BphC_JF8 protein was modeled in the tetrameric state using the crystal structure of catechol 2,3-dioxygenase (MPC; Protein Data Bank code 1MPY [PDB] ) as the template (4). A preliminary model of the BphC_JF8 was constructed using automated modeling servers SWISS-MODEL (38) and FAMS (39). The final model was derived with the program package MODELLER version 6.0 (40) and optimized by "refine1" molecular dynamic simulation as implemented in the same package. The stereochemical quality of the final model was assessed by the program PROCHECK version 3.0 (41). The presence of salt bridges was inferred when Asp or Glu side chain carbonyl oxygen atoms were found to be within a 4.0-Å distance from the nitrogen atoms in Arg and Lys side chains.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis of the Cloned meta-Cleavage Gene—A 4-kb HindIII fragment was isolated from the transformant (pBHC1), which could convert 2,3-dihydroxybiphenyl into the yellow meta-cleavage product. Restriction analysis and sub-cloning resulted in the identification of a 1.5-kb HindIII-SacI fragment (pBH1) that encoded the extradiol dioxygenase activity. Sequencing the 1.5-kb fragment revealed a 945-bp open reading frame with a G + C content of 48%. The C-terminal region of an open reading frame upstream of the meta-cleavage gene exhibited homology to known dihydrodiol dehydrogenase genes (bphB), implying that the meta-cleavage gene is not isolated but is most probably part of an operon. The 16 S rDNA sequence (97.8% identity to Bacillus stearothermophilus) and various chemotaxonomic markers indicate that strain JF8 is related to B. stearothermophilus (24). Therefore, the codon usage of the bphC gene was compared with that of B. stearothermophilus and was found to be similar with one exception. The codon CCC, which is very rarely used in B. stearothermophilus, was found to be most frequently used to code for proline in BphC_JF8.

Purification of the Biphenyl-induced Extradiol Dioxygenase and Its N-terminal Sequencing—Initially, the inducible extradiol dioxygenase from biphenyl-grown cells of Bacillus sp. JF8 was purified. The purification scheme for the native enzyme is given in Table I, and the enzyme was purified 32-fold with an overall yield of 54%. The SDS-PAGE of the purified enzyme showed one distinct band (Fig. 2). The N-terminal sequence of the native enzyme was determined by Edman degradation to be TAEIAKFGHIALITPNLEKSVWFFRDIVGLEEVDRQGDTI. This agrees with the DNA sequence data, except for the initial Met, of the cloned open reading frame, identifying the extradiol dioxygenase gene as a bphC. The bphC gene of JF8 was expressed from pQW1 in E. coli, and a three-step protocol (similar to one shown in Table I) was used to purify the recombinant extradiol dioxygenase, giving a 19-fold purification with a total recovery of 46%. The determined N-terminal sequence of the recombinant enzyme was identical to that of the native enzyme.


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  		TABLE I
Purification of the native 2,3-dihydroxybiphenyl 1,2-dioxygenase of strain JF8

 


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  		FIG. 2.
SDS-PAGE of purified 2,3-dihydroxybiphenyl 1,2-dioxygenase. Lane 1, molecular mass standards; lane 2, 2.5 µg of 2,3-dihydroxybiphenyl 1,2-dioxygenase recombinant protein purified from E. coli; lane 3, 1.0 µg of 2,3-dihydroxybiphenyl 1,2-dioxygenase purified from Bacillus sp. JF8.

 

Metal Analysis of BphC_JF8 —The recombinant BphC_JF8 grown on Luria broth had very low specific activity (0.68 units/mg) compared with the native enzyme (5.47 units/mg), and we tried to activate the recombinant enzyme in the presence of Fe(II) and Mn(II). Activation in the presence of 1 mM Mn(II) at 25 and 60 °C is shown in Fig. 3. Activation at 60 °C was faster and resulted in a higher specific activity as compared with 25 °C. When the enzyme was incubated with 1 mM Fe(II) and ascorbate in argon gas, the enzyme was activated 20-fold. However, the activity diminished rapidly and came down to the original level in 20 s (results not shown).



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  		FIG. 3.
Activation of purified, recombinant BphC_JF8. Enzymes were dialyzed against 50 mM Tris-HCl buffer (pH 7.5) and incubated in the presence of 1 mM Mn2+ at 25 and 60 °C.

 

Metal analysis using ICP-MS showed that the native Bph-C_JF8 contained between 4.0 and 4.8 manganese atoms per enzyme molecule, depending on the batch. The iron content was found to be consistently low at 0.05 iron atoms/enzyme molecule. The oxidation state of the manganese in BphC_JF8 was determined using EPR spectroscopy (Fig. 4A). The typical 6-fold signal centered at g = 2.02 clearly showed the presence of Mn(II). The hyperfine coupling constant, A, of 9.3 mT compares well with that observed for other Mn(II)-dependent enzymes (42–44). An unusual feature of the BphC_JF8 spectrum was the presence of an intense six-line signal at g = 4.06, which had an A value of 9.0 mT. An additional signal was also observed around g = 4.8, which overlapped with the peaks at g = 4.06. When the substrate, 2,3-dihydroxybiphenyl, was added to BphC_JF8 under anaerobic conditions, the EPR spectrum changed dramatically (Fig. 4B). The intense signal at g = 4.06 and g = 4.8 disappeared, and the signal at g = 2.02 increased in intensity.



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  		FIG. 4.
X-band EPR spectra of 0.15 mM BphC_JF8 (A) and 0.05 mM BphC_JF8 plus 0.5 mM 2,3-dihydroxybiphenyl (anaerobic) (B) in 50 mM potassium phosphate buffer (pH 7.5) at 10 K. Both spectra are normalized to correct for differences in enzyme concentration and instrumental gain.

 

Biochemical Characterization and Stability of the BphC Enzyme—SDS-PAGE analysis showed that the purified enzyme had an apparent molecular mass of 35 kDa, which is in good agreement with the value calculated from the deduced amino acid sequence of the enzyme. The native molecular mass of the enzyme was estimated to be 125 ± 10 kDa by gel filtration, indicating that the native enzyme is a homotetramer. To determine the substrate specificity, the enzyme was tested for its ability to oxidize 2,3-dihydroxybiphenyl, catechol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol. The Km and Vmax values are listed in Table II. The Km for 2,3-dihydroxybiphenyl was found to be lower, almost 1/10 that for 3-methylcatechol and 1/1084 that for catechol, implying a better fit of the enzyme with the bigger substrate. The Km values with 2,3-dihydroxybiphenyl as substrate were determined at two different temperatures, and a decrease in the Km was observed at the higher temperature (Table III). BphC_JF8 showed a maximum specific activity of 5.47 µmol/mg of protein at 60 °C, which is low compared with other 2,3-dihydroxybiphenyl 1,2-dioxygenases (i.e. Pseudomonas pseudoalcaligenes KF707, 87.2 µmol/mg of protein (45); Burkholderia cepacia LB400, 191 µmol/mg of protein (46); and even strain BN6 at 7.0 µmol/mg of protein (47)). BphC_JF8 exhibited a higher affinity for its substrate, as evidenced by a Km of 0.095 µM, compared with BphC_KF707 (87 µM) (45) and BphC_LB400 (7 µM) (46). The high affinity exhibited by the enzyme for 2,3-dihydroxybiphenyl indicates that it can efficiently utilize low ambient concentrations of the substrate. Except for the specific activity, the native and recombinant enzyme exhibited identical molecular weight and other biochemical characteristics such as substrate specificity, Km values, temperature, and pH optimum.


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  		TABLE II
Kinetic parameters of 2,3-dihydroxybiphenyl 1,2-dioxygenase cloned from Bacillus sp. JF8

 

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  		TABLE III
Kinetic parameters of 2,3-dihydroxybiphenyl 1,2-dioxygenases with 2,3-dihydroxybiphenyl as substrate

 

The activation energy for the meta-cleavage of 2,3-dihydroxybiphenyl and catechol by BphC_JF8, BphC of Rhodococcus sp. RHA1 (BphC_RHA1) and XylE of the TOL plasmid, pWWO (MPC_mt2), was determined from the linear range of the Arrhenius plot. The activation energy for the meta-cleavage of 2,3-dihydroxybiphenyl by BphC_JF8 was determined to be 14.5 kcal/mol, whereas for catechol the activation energy was 9.9 kcal/mol. In contrast, the activation energy for the meta-cleavage of 2,3-dihydroxybiphenyl and catechol by Bph-C_RHA1 was 8.4 kcal/mol and 6.9 kcal/mol, respectively, whereas for MPC_mt2, the activation energy for 2,3-dihydroxybiphenyl was 10.4 kcal/mol, and for catechol, it was 8.1 kcal/mol.

The optimal temperature and pH for BphC_JF8 activity were examined. The enzyme was most active at 85 °C and pH 7.5 under standard assay conditions. The thermostability of the enzyme was examined by measuring the remaining activity after incubation at various temperatures. The enzyme retained 100% of its activity after treatment at 60 °C and 75% of its activity after treatment at 70 °C for 60 min. The thermostability of the enzyme at 70, 75, and 80 °C is shown in Fig. 5.



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  		FIG. 5.
Thermostability of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Bacillus sp. JF8. Residual activities were assayed after preincubation of 0.4 mg of enzyme/ml at 70 °C ({circ}), 75 °C ({triangleup}), and 80 °C (•).

 

BphC_JF8 did not display substrate inhibition even at 3 mM 2,3-dihydroxybiphenyl and was not inhibited at 0.5 mM 3-chlorocatechol. After prolonged incubation (60 min at 25 °C), the purified enzyme was only partially inhibited at high concentrations of Fe(III) ions, with 5 mM Fe(III) causing a 35% inhibition. The enzyme was also resistant to inactivation by various chelators. Incubation in 5 mM EDTA, 1,10-phenanthroline, 2,2'-bipyridyl, and Tiron for 60 min (25 °C) resulted in almost no inhibition (95–100% residual activity). Incubating the enzyme in 25 mM EDTA for 90 min resulted in a gradual loss of activity (87% residual activity after 60 min of incubation and 81% residual activity after 90 min of incubation), whereas 25 mM 1,10-phenanthroline and 200 mM NaF for 60 min did not inhibit the enzyme. Incubation with 0.1 mM H2O2 for 60 min did not inhibit the enzyme, although 1 mM H2O2 for 60 min resulted in weak inhibition (86% residual activity).

Homology to Other Extradiol Dioxygenases—The deduced amino acid sequence of BphC_JF8 was compared with other Type I extradiol dioxygenases. The gene product has no more than 38% identity with known extradiol dioxygenases, and in a phylogenetic tree, BphC_JF8, which exhibits less than 25% identity with 2,3-dihydroxybiphenyl 1,2-dioxygenases cloned from biphenyl/PCB-degrading organisms, clusters with enzymes that cleave monocyclic compounds, specifically with the catechol 2,3-dioxygenases from R. rhodochrous CTM, Cdo_CTM (48), and B. stearothermophilus FDTP-3, PheB_FDTP3 (49).

Conservation of Catalytically Important Residues—In BphC_JF8, amino acid residues that (i) define the substrate binding pocket, (ii) play a role in the folding and tertiary structure of known dioxygenases, and (iii) are a part of the extradiol dioxygenase fingerprint region are well conserved. On aligning 23 extradiol dioxygenases, Eltis and Bolin (50) found nine strictly conserved residues. These are the metal ligands His-146, His-210, and Glu-260 (numbering as in BphC_LB400); the active site residues His-195, His-241, and Tyr-250; and Gly-28, Leu-165, and Pro-254. The last three residues are remote from the active sites but are located near the interface between the N- and C-terminal domains and probably play a structural or folding role (50). In BphC_JF8, eight of the above mentioned residues are conserved (Fig. 6), Leu-165 being replaced with a Met residue. Phe-187 of BphC_LB400, which lines the substrate binding pocket may be involved in a weakly polar interaction with the substrate, and this interaction is conserved in the enzyme-substrate complex of all extradiol dioxygenases, with Trp (as observed in BphC_JF8) or His replacing Phe in some enzymes. Two other residues that line the binding pocket of the hydroxylated ring in BphC_LB400 are Asn-243 and Asp-244, with the amide group of Asn-243 interacting weakly with the ring of Phe-187. The Asn-Asp sequence was found exclusively in two-domain enzymes that preferentially cleave bicyclic substrates (50). Although BphC_JF8 preferentially cleaves a biphenyl ring, the Asn-Asp residues are replaced by Ile-Ser residues as in other dioxygenases that preferentially cleave monocyclic compounds. Val-148 of BphC_LB400, which interacts directly with the distal ring of 2,3-dihydroxybiphenyl, is replaced by an Asn residue in BphC_JF8.



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  		FIG. 6.
Structural comparison of extradiol dioxygenases. Protein sequence alignment of BphC_JF8 with the catechol 2,3-dioxygenase (metapyrocatechase) of P. putida mt-2 (MPC_mt2) and 2,3-dihydroxybiphenyl 1,2-dioxygenase of Pseudomonas sp. KKS102 (BphC_KKS102) and B. cepacia LB400 (BphC_LB400). Numbering of amino acids in BphC_JF8 and BphC_LB400 is indicated above and below the alignment, respectively. Amino acids common in at least three sequences are shaded in gray, whereas those common in all four sequences are boxed in gray. *, metal ligands; +, active site residues; #, residues that have been found to be conserved in most dioxygenases and are presumed to play a structural or folding role. The known secondary structure elements for MPC and BphC_LB400 are indicated below the alignment, whereas the predicted secondary structure elements for BphC_JF8, using PSI-Pred (35), Jpred (36), PHD (37), and SSPro (38) methods, are indicated as rectangles above the alignment, with {beta}-strands shown in black and helices shown in gray. In the secondary structure of MPC and BphC, {beta}-strands are shown as arrows, whereas helices are shown as gray rectangles.

 

Conservation of Secondary Elements and Protein Fold—Secondary structure predictions from the deduced amino acid sequence of BphC_JF8 using different methods returned highly consistent results. Some of the elements (the second {alpha}-helices of the N- and C-terminal domains, the second {beta}-strand of the N-terminal domain, and the fifth, seventh, and eighth {beta}-strand of the C-domains) were predicted with very high certainty, including the beginning and ends of the elements (Fig. 6). The distribution of the secondary elements along the sequence of BphC_JF8 was very similar to that observed in MPC_mt2 (4), with a few exceptions. The modeled structure for BphC_JF8 suggests that the monomer has N- and C-terminal domains that are structurally similar, and each domain is composed of an eight-stranded half-opened {beta}-barrel as observed in MPC_mt2 (4), BphC_LB400 (2), and BphC_KKS102 (3). The N- and C-terminal domains of BphC_JF8 have eight {beta}-strands and two {alpha}-helices; however, the secondary structure of the last 26 residues in the C-terminal domain of BphC_JF8 is not well defined in relation to MPC_mt2. In this region, MPC_mt2 has one strand and two helices that appear to be absent in the BphC_JF8 protein. The long loop present between the N- and C-terminal domains in MPC_mt2 is also present in BphC_JF8.

Salt bridges may play a role in resisting unfolding of protein structures at elevated temperatures, since the number of salt bridges has been observed to be higher in thermophilic proteins than in their mesophilic homologues. The number of intersub-unit salt bridges in BphC_JF8 was compared with MPC_mt2 (Table IV). 40 intersubunit salt bridges were detected in the model of BphC_JF8, which is significantly higher than the 28 bridges present in the structurally similar but mesophilic protein MPC_mt2.


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  		TABLE IV
Comparison of intrasubunit salt bridges identified in the predicted structure of BphC_JF8 and crystal structure of MPC_mt2

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have cloned and characterized a novel Mn(II)-dependent BphC that besides being thermostable is also resistant to many chelators/inhibitors. The N-terminal amino acid sequence of the extradiol dioxygenase induced when strain JF8 was grown on biphenyl is identical to the deduced amino acid sequence of the cloned gene, and the substrate preference and affinity of BphC_JF8 for 2,3-dihydroxybiphenyl establishes and verifies the role of the enzyme.

To facilitate comparison, Vieille et al. (51) had proposed that thermostability, which is the capacity of the enzyme to resist irreversible thermal inactivation, be expressed as the temperature at which the enzyme half-life is 1 h. In the case of BphC_JF8, the value lies between 75 °C (half-life of 81 min) and 80 °C (half-life 30 min). Available reports indicate that the BphC of Pseudomonas putida OU83 (which exhibits 94% identity to BphC_LB400) lost 47% of its activity at 37 °C and all activity at 65 °C (52), whereas BphCII from Rhodococcus globerulus P6 retained only 10% activity after 10 min of incubation at 50 °C (53). In comparison, the half-life of BphC_JF8 at 75 °C was 81 min, indicating that it is probably the most thermostable 2,3-dihydroxybiphenyl 1,2-dioxygenase isolated. The lowest Km value for the substrate was at the physiological growth temperature (Km of 0.35 µM for 2,3-dihydroxybiphenyl at 25 °C compared with 0.095 µM at 60 °C), as has been observed in enzymes from psychrophiles (54) and thermophiles (55).

The Ea values of reactions catalyzed by enzymes from heat-adapted organisms are usually higher than those catalyzed by corresponding enzymes from mesophiles. On comparing the Ea for oxygenation of 2,3-dihydroxybiphenyl and catechol by Bph-C_JF8 and two mesophilic counterparts, BphC_RHA1 and MPC_mt2, the Ea value of BphC_JF8 for both substrates was found to be higher, indicating that BphC_JF8 is adapted for functioning at higher temperatures.

In the classification of extradiol dioxygenases by Eltis and Bolin (50), based on phylogenetic consideration, enzymes belong to a superfamily that is divided into families and several subfamilies, with sequences within the same subfamily exhibiting >54% identity. BphC_JF8 exhibits a 36% identity (62% similarity) with Cdo_CTM and 38% identity (63% similarity) with PheB_FDTP3. Clearly, the thermostable BphC_JF8 belongs to a new subfamily.

Results of ICP-MS indicate that BphC_JF8, a homotetramer, is fully occupied with 4 gram atoms of manganese, indicating a stoichiometric metal content, whereas MndD_CM2 had 2.7 to 3.6 gram atoms of manganese/homotetramer (42). In the EPR spectrum of BphC_JF8, a 6-fold hyperfine splitting signal at g = 2.02 clearly indicated the presence of Mn(II) (nuclear spin, I = 5/2). The intense signals at g = 4.06 and g = 4.8 are unusual. Human manganese superoxide dismutase has a metal ion coordinated by five ligands (four protein ligands and one water molecule) (56), and a site-specific mutant (Q143N) of the enzyme (Q143N), which has a predominance of Mn(II) in the resting state, exhibits a spectrum with hyperfine splitting between 100 and 200 mT (57), which looks very similar to the lower field signals of BphC_JF8. Probably, the coordination of the ligands to the active site Mn(II) in BphC_JF8 and Q143N human manganese superoxide dismutase are similar, although the enzymes have differing coordinating residues. In some Mn(II)-dependent enzymes where signals at g = 4 are observed (e.g. oxalate decarboxylase of B. subtilis (g = 4) (44) and oxalate oxidase of barley (g = 4.4) (43)), the relative intensity of the signal at g = 4 is about 10–100-fold lower than the signal at g = 2. In BphC_JF8, the intense g = 4.06 signal may be caused by zero-field splitting derived from strong axial perturbation due to five-coordinate Mn(II) as reported in reduced manganese-superoxide dismutase from E. coli (58). The spectrum of bound Mn(II) is remarkably sensitive to structural changes that occur upon binding of substrate (59), and in MndD_CM2, a 6-fold signal at g = 4.3 arises only when the enzyme is anaerobically exposed to its natural substrate (43), whereas in BphC_JF8, the opposite phenomenon is observed. Binding of the substrate, 2,3-dihydroxybiphenyl, to BphC_JF8 under anaerobic conditions resulted in elimination of the intense signals at g = 4.06 and g = 4.8 observed in the resting state of the enzyme, probably due to coordination of substrate to the Mn(II) center.

BphC is subject to two forms of substrate inhibition, reversible substrate inhibition and mechanism-based inactivation or suicide inhibition (60). Reversible substrate inhibition has been reported in a number of 2,3-dihydroxybiphenyl 1,2-dioxygenases (18, 28). Suicide inhibition by the preferred substrate has been reported for both catechol 2,3-dioxygenase (61) and BphC (60), although BphC is more susceptible to inactivation. However, BphC_JF8 did not exhibit substrate inhibition even at 3 mM 2,3-dihydroxybiphenyl. 3-Chlorocatechol has also been reported to be a potent suicide inhibitor of the BphC enzymes. Vaillancourt et al. (62) show that inactivation of the enzyme does not involve covalent modification or hydroxylation of an active site residue but arises principally from the oxidation of the active site Fe(II) to Fe(III). BphC_JF8, which has an active site Mn(II), is not inhibited by 0.5 mM 3-chlorocatechol.

A homology three-dimensional model of BphC_JF8 was constructed using MPC_mt2 (4) as a template (results not shown). The crystal structures of three extradiol dioxygenases, Bph-C_LB400 (2), BphC_KKS102 (3), and MPC_mt2 (4), indicate that the monomer consists of two domains containing repetition of an {alpha}{beta} module ({beta}1{alpha}{beta}2{beta}3{beta}4). Both the BphC enzymes are octamers, whereas the MPC_mt2 is a tetramer. In the end region of the N-terminal domain of MPC_mt2, there is a long protruding loop region that prevents the formation of an octameric structure (4). In the model of BphC_JF8, a similar loop is present between the last {beta}-sheet of the N-terminal domain and the first {beta}-sheet of the C-terminal domain (Fig. 6). This structural feature could explain the tetrameric structure of the BphC_JF8. In the C-terminal domain of MPC_mt2, a region consisting of one strand and two helices follows the last {beta}{alpha}{beta}{beta}{beta} motif, and the last helix is located so as to cover and narrow the open region of the {beta}-barrel, which is the substrate entrance site into the active site located in the C-terminal domain (4). These last two helices are not conserved in BphC_JF8, indicating that the tunnel entrance could be bigger, and this could play an important role in determining the substrate specificity of the enzyme, which is very different from MPC_mt2.

Although the metal ligands and catalytic residues are conserved, a strictly conserved residue (Leu-165 of BphC_LB400 and BphC_KKS102), which could play a structural or folding role, and several other residues associated with enzymes preferentially cleaving bicyclic compounds are not conserved in BphC_JF8. From the predicted structure of BphC_JF8, it appears that replacement of the conserved Leu by a hydrophobic Met residue would not result in a significant change because the increased size of the side chain due to the Leu -> Met substitution is compensated by a substitution in the residue that is in van der Waals contact with Met-172 (Leu-168 of BphC_JF8), which is in a position corresponding to Tyr-161 of BphC_LB400 and BphC_KKS102. Substitution of Phe-187 of BphC_LB400 or BphC_KKS102, a residue that directly interacts with the substrate bound in the active site of the enzyme, with Trp-191 of BphC_JF8 could influence substrate specificity. More importantly, we find that in BphC_JF8, the presence of Trp-191 along with the substitution of an Asn residue (Asn-155) in the position of Val-148 of BphC_LB400 or BphC_ KKS102 results in reducing the volume of the putative "oxygen binding cavity" identified by Senda et al. (3), although a smaller Gly residue (Gly-202) is substituted for Ala-198 of BphC_ LB400. Of the two cavities identified for O2 binding, Senda et al. (3) believed that the oxygen binding cavity was more suitable, since the best attack on the C-1 carbon of the substrate by an oxygen atom could be performed at this site. The other putative site for O2 binding was identified between the iron ion and Asp-243 of BphC_KKS102. In BphC_JF8, this site is larger due to the substitution of Ser-252 instead of an Asp residue. However, binding of dioxygen to this site would require a different orientation of the biphenyl molecule than observed in the enzyme-substrate complex of BphC_KKS102 (structure Protein Data Bank ID code 1EIM [PDB] ), to allow for a direct attack on the C-1 atom of the substrate by the dioxygen molecule. Therefore, binding of the dioxygen to the oxygen binding cavity appears to be more likely; however, the dioxygen would be positioned closer to the substrate molecule, since the cavity is smaller. We speculate that the smaller size of the cavity is responsible for the resistance of BphC_JF8 to denaturation by H2O2, since it would exclude the molecule from binding in the cavity. This perturbation in the O2 binding site might also explain the poor stability of the Fe(II) form of BphC_JF8, probably making the iron ion more susceptible to uncoupled oxidation and inactivation in the presence or absence of substrate.

It is expected that residues involved in the interaction with the substituted phenyl moiety of 2,3-dihydroxybiphenyl would be replaced with larger residues in those extradiol dioxygenases that preferentially cleave monocyclic compounds. Eltis and Bolin (50) noted that Val-148 of BphC_LB400, which interacts directly with the distal ring of 2,3-dihydroxybiphenyl, tends to be substituted by larger residues in catechol 2,3-dioxygenase (Leu-155, MPC_mt2). In BphC_JF8, this residue is replaced by Asn (as in PheB_FDTP3 and Cdo_CTM). In the predicted model of BphC_JF8, the presence of an Asn residue is sterically similar to the Leu residue of MPC_mt2; however, electronically it may be significant due to the different partial charges on the nitrogen and oxygen atoms of the Asn residue. Furthermore, Asn can donate and accept hydrogen bonds, which is not possible for Leu of MPC_mt2 or Val of BphC_LB400.

Although salt bridges appear to make little contribution to protein stability at room temperature, they could play a crucial role in promoting thermostability in protein (63). In a statistical examination of factors enhancing protein thermostability, Kumar et al. (64) found that salt bridges were the only structural features that showed a consistent increase with thermal stability of proteins. The role of salt bridges in stabilization was also inferred from comparative studies on glutamate dehydrogenase from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis and the mesophile Clostridium symbiosum (65, 66). Disruptive mutational analysis has been used in citrate synthase from P. furiosus (67) to underline the importance of salt bridges on thermostability. It is possible that the large number of intersubunit salt bridges in BphC_JF8 (Table IV) play a role in its thermostabilization.

The proline rule for thermostabilizing proteins had been proposed by Suzuki (68, 69). PheB_FDTP-3 is thermostable (49), whereas the stability of PheB from Bacillus thermoleovorans A2 (PheB_A2) (70) is comparable with that of mesophilic enzymes. Comparing these proteins with BphC_JF8 indicates that Pro residues may play a role in its thermostabilization. The percentage of Pro residues in PheB_A2 is only 4.3% compared with 6.7% and 6.4% for BphC_JF8 and PheB_FDTP3, respectively. Indeed, the number of Pro residues was found to be higher in thermostable extradiol dioxygenases compared with their mesophilic counterparts (BphCI_P6, 4.12%; BphC_RHA1, 2.83%; BphC_KKS102, 5.46%; BphC_ LB400, 3.67%; Cdo_CTM, 5.91%; MPC_mt2, 3.9%) with one exception, MndD_CM2 (7.28%).

Sequencing MndD_CM2 indicated that the catalytically active residues that are conserved in all Fe(II)-dependent extradiol dioxygenases are also conserved in MndD (6). Boldt et al. (71) showed that the conserved His (His155 and His214) and Glu (Glu266) residues of MndD_CM2 act as ligands to Mn(II). As observed in superoxide dismutase (72), the extradiol dioxygenases appear to utilize identical coordinating residues for their Fe(II)- and Mn(II)-dependent enzymes. We assume that in BphC_JF8, the analogous His-153, His-216, and Glu-269 act as the Mn(II) coordinating residues. In the catalytic mechanism proposed for Fe(II) extradiol dioxygenases (3, 73), the substrate binds to the active site Fe(II), followed by O2 binding to the Fe(II) and the activated iron-bound dioxygen subsequently attacking at C-1 of the substrate. It is possible that a Mn(II) extradiol dioxygenase could function in an analogous manner; however, the resolution of our homology model and also the different electronic configurations of Fe(II) compared with Mn(II) do not allow a reliable proposal of the reaction mechanism. In superoxide dismutase, although it was assumed that the iron-superoxide dismutase mechanism would apply to manganese-superoxide dismutase as well, a close look at the stringent metal specificities of the enzyme uncovered several subtle differences between iron-superoxide dismutase and manganese-superoxide dismutase (74, 75) due to the intrinsic differences in the reactivities of the metals. Further work is necessary to elucidate the electronic configuration and structural reasons that lead some extradiol dioxygenases to utilize iron and others to utilize manganese.

Phylogenetic analysis of MndD_CM2 had placed the protein with the single ring substrate subfamily of the extradiol dioxygenase. This had led Whiting et al. (42) to propose a common ancestor for the Fe(II)- and Mn(II)-dependent extradiol dioxygenases. While studying the mechanism for metalloprotein evolution, Bergdoll et al. (76) noted that although bleomycin resistance protein from Streptoalloteichus hindustanus, BphC_LB400, and human glyoxales I possess less than 20% sequence identity, three-dimensional structure of the bleomycin resistance protein and glyoxales I dimers and BphC monomers are similar, with four superimposable copies of the {beta}{alpha}{beta}{beta}{beta} modules and active sites in similar location and structure. They hypothesize that the ability to bind a metal ion via four ligands was a crucial step in the evolution of extradiol dioxygenases. We can only speculate on whether the ancestral gene for extradiol dioxygenase was Fe(II)- or Mn(II)-dependent or whether the ancestral gene was able to accommodate both the metal ions as has been observed for the cambialistic superoxide dismutase (77). Most of the extradiol dioxygenases isolated so far are Fe(II)-dependent dioxygenase, which would appear to indicate that iron is the preferred metal ion; however, the ability of BphC_JF8 to withstand conditions that inhibit Fe(II)-dependent extradiol dioxygenases indicates the advantage of Mn(II)-dependent enzymes.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB092521 [GenBank] .

* This work was supported in part by Grant-in-aid for Scientific Research 12660091 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Takashi Hatta Research Institute of Technology, Okayama University of Science, 401-1 Seki, Okayama 703-8232, Japan. Tel.: 81-86-278-9349; Fax: 81-278-5312; E-mail: thatta{at}po.harenet.ne.jp.

1 The abbreviations used are: PCB, polychlorinated biphenyl(s); X-gal, 5-bromo-4-chloro-3-indolyl-{beta}-D-thiogalactopyranoside; ICP-MS, inductively coupled plasma mass spectrometry; mT, millitesla. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Shin Toyoda (Okayama University of Science) and Drs. Toshiki Masumizu and Yukio Mizuta (JEOL Ltd.) for analysis of the EPR. We also thank Dr. Jun Naohara (Okayama University of Science) for the ICP-MS analysis. We appreciate the discussions on coordination state of metal ions in the structure of BphC_JF8 with Dr. Petr Kulhanek (National Centre for Biomolecular Research, Masaryk University, Brno). We are grateful for the constructive comments of two anonymous reviewers.



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