1 Department of Built Environment, Tokyo Institute of Technology, Yokohama 226-8502, Japan
2 Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38, Hikari-cho, Kokubunji, Tokyo 185-8540, Japan
3 Research Institute of Technology, Okayama University of Science, Okayama 703-8232, Japan
Correspondence
Kazuhide Kimbara
kimbara{at}rtri.or.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB116258.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The degradation of aromatic compounds by thermophilic organisms and thermostable enzymes, especially when applied in biotechnology processes, could provide important advantages compared to mesophiles. The elevated temperatures would reduce the risk of contamination, and the bioavailability of the hydrophobic contaminants would increase. The improved stability of thermophilic enzymes compared to their mesophilic homologues is well documented (Sterner & Liebl, 2001; Vieille et al., 1996
). Although thermophiles which can degrade compounds such as BTEX (benzene, toluene, ethylbenzene, xylene), phenol, cresol and naphthalene have been reported (Chen & Taylor, 1995
; Buswell, 1974
, 1975
; Duffner & Muller, 1998
; Duffner et al., 2000
; Shimura et al., 1999
), the metabolic pathways involved in the degradation of aromatic compounds have not been well studied in these organisms and there is very little information on the genes and proteins involved.
Bacillus sp. JF8 is a thermophilic polychlorinated biphenyl (PCB) degrader which can utilize biphenyl and naphthalene as the sole carbon and energy source (Shimura et al., 1999). The presence of separate pathways for the degradation of biphenyl and naphthalene was indicated by Bacillus sp. JF8N, a mutant of Bacillus sp. JF8 which had lost the ability to utilize biphenyl as a carbon source while retaining the ability to use naphthalene (Shimura et al., 1999
). Further studies indicated that Bacillus sp. JF8 possessed a 40 kb plasmid which was absent in the mutant JF8N, hinting that the bph genes were borne on the plasmid while the nah genes were located on the chromosome (G. Mukerjee-Dhar, M. Shimura, T. Hatta & K. Kimbara, unpublished data). Hatta et al. (2003)
isolated and characterized an atypical Mn(II)-dependant extradiol dioxygenase, BphC_JF8, which was encoded on the plasmid.
Here we report the characterization of the two extradiol dioxygenases from Bacillus sp. JF8, both induced by naphthalene; one is thermostable and Mn(II)-dependent while the other is more thermolabile and Fe(II)-dependent. To the best of our knowledge, this is the first time Mn(II)- and Fe(II)-dependent extradiol dioxygenases have been isolated from the same organism and found to be transcribed in the same operon.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Southern hybridization.
A DNA probe, spanning the determined N-terminal region of the extradiol dioxygenase that was isolated from naphthalene-grown Bacillus sp. JF8, was generated using PCR with the primers C1-F (5'-GGCCATGCCGAACTTTTTGTCA-3') and C1-R (5'-TCTTTATCAGCATCGTTTTCGCG-3'). The probe was labelled with digoxigenin (DIG; Roche). Total DNA of Bacillus sp. JF8N was digested with BamHI, ClaI, EcoRI, HindIII and SalI, and fractionated by electrophoresis in 0·7 % agarose gel before transfer to Hybond-N+ nylon membranes (Amersham). For colony hybridization, the recombinant E. coli colonies were transferred to Hybond-N+ nylon membranes. Southern hybridization was performed at 60 °C with high-stringency washes at 60 °C (2x15 min, 0·1xSSC+0·1 % SDS).
Cloning and DNA sequencing.
SalI and BamHI gene banks (5 kb and 2 kb, respectively) of the chromosomal DNA of Bacillus sp. JF8N were constructed in pUC18 (Takara Biomedicals) and recombinant E. coli colonies probed with the DIG probe mentioned above. A clone containing a 5 kb SalI fragment designated pCSl5 and a clone containing a 2 kb BamHI fragment designated pCBm2 gave a positive hybridization signal. DNA regions upstream of the 5 kb SalI fragment and downstream of the 2 kb BamHI fragment were isolated by inverse PCR as described elsewhere (Innis et al., 1990). PCR reactions were carried out with the primers up-F (5'-ACGATCATCGATCTGCTGG-3'), up-R (5'-AACACTTCTTCCGTTCGTGG-3') (for upstream), dw-F (5'-GGAACTCTTCGGACACC-3'), dw-R (5'-CGCAATGCATAGTCGACG-3') (for downstream). Conditions for amplification of the upstream region by inverse PCR were: 35 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, followed by cooling to 4 °C. Conditions for amplification of the downstream region were: 35 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 3 min, followed by cooling to 4 °C. SphI-digested JF8N genomic DNA was used as template to isolate a 2 kb DNA fragment upstream of pCSl5 while EcoRI-digested DNA was used to isolate a 3 kb fragment downstream of pCBm2. Deletion mutants were constructed by the Kilo-sequence Deletion kit (Takara), using the exo/mung system. Nucleotide sequences were determined using the CEQ DTCS-Quick Start Kit (Beckman Coulter) by the dideoxy-chain-termination method, with a CEQ2000 DNA sequencer (Beckman Coulter).
Sequence analysis.
DNA sequences were analysed by GENETYX software (Genetyx, Japan) and the deduced ORFs compared to those in the databases using BLAST and FASTA programs. Amino acid sequences exhibiting homology were retrieved from the protein database and aligned using CLUSTAL W (Thompson et al., 1994). A phylogenetic tree was constructed using the neighbour-joining method and depicted using TreeView software (Page, 1996
).
Purification of the native and recombinant extradiol dioxygenases.
Bacillus sp. JF8 was grown on Castenholz D agar plates in the presence of naphthalene vapour. The cells were harvested, washed and resuspended in 20 mM phosphate buffer (pH 7·5) containing 2 mM -mercaptoethanol (buffer A). The cell suspension was passed through a French press (Thermo IEC) and centrifuged at 17 000 g for 60 min and the supernatant applied to a DEAE-Toyopearl 650M (Tosoh, Japan) column. The enzyme was eluted with a 4000 ml gradient of 0·00·4 M KCl. The enzymic activities of the eluted fractions were assayed against 2,3-dihydroxybiphenyl (as described below). The active fractions were collected, dialysed, and applied to a Phenyl Sepharose HP 26/10 column (Amersham) equilibrated with buffer A containing 1 M ammonium sulfate. The enzyme was eluted with a 400 ml gradient of 1·00·0 M ammonium sulfate. The enzymic activities of the eluted fractions were assayed against 2,3-dihydroxybiphenyl (as described below). The active fractions were collected and dialysed against buffer A and applied to a Mono Q HR 16/10 column (Amersham) equilibrated with buffer A. The enzyme was eluted with a 400 ml gradient of 0·10·35 M KCl. The fractions containing enzyme activity were pooled and dialysed against buffer A. The N-terminal sequence of the native enzyme was determined by automated Edman degradation on a model 492 protein sequencer (Applied Biosystems).
E. coli cells containing pCSP2 [2 kb SalIPstI fragment encoding nahH, the C-terminal region of tnp1, and part of nahL in pBluescript II KS (+) (Stratagene)] and pCBm2 (2 kb BamHI fragment encoding mocB and nahC in pUC18) were cultivated at 37 °C in the presence of 1 mM IPTG in 400 ml LB medium containing 100 µg ampicillin ml1 (see Fig. 1 for plasmids). Harvested cells of the recombinant E. coli were suspended in buffer A, and sonicated with a homogenizer (Subsonic HMO-100; Iwaki, Japan) for 3 min. Cell debris was removed by centrifugation for 30 min at 18 000 g. The supernatant was referred to as the cell-free extract.
|
Electrophoresis and activity staining of extradiol dioxygenases.
SDS-PAGE was carried out according to the method of Laemmli (1970) with Prestained Protein Marker Broad Range (New England Biolabs) and native nondenaturing PAGE was performed with the HMW Native Marker Kit (Amersham) using the same solutions without SDS. PAGE studies were done with purified recombinant NahC_JF8 and recombinant NahH_JF8 in cell-free extract. After electrophoresis, the native gels were assayed for extradiol dioxygenase activity by incubating the gels in 100 mM Tris/HCl (pH 8·0), 0·5 mM
-mercaptoethanol solution containing 2,3-dihydroxybiphenyl. Visual observation indicated formation of the yellow meta-cleavage product. Gels were stained with Coomassie brilliant blue R250 (Sambrook et al., 1989
). The relative molecular masses were calculated from the mobilities of the marker proteins.
Enzymic assays.
Extradiol dioxygenase activity was estimated by following the formation of the ring-fission products from the corresponding substrates using a DU-650 spectrophotometer (Beckman Coulter) at 25 °C. Reaction mixtures contained 50 mM sodium phosphate buffer (pH 7·5), substrate and enzyme solution. The absorption coefficients used for the ring-fission products of the substrates were as follows: catechol, max 375 nm,
33 mM1 cm1 (Bayly et al., 1966
); 3-methylcatechol,
max 388 nm,
13·8 mM1 cm1; 4-methylcatechol,
max 82 nm,
17 mM1 cm1; 2,3-dihydroxybiphenyl,
max 434 nm,
13·2 mM1 cm1; 4-chlorocatechol,
max 379 nm,
40 mM1 cm1 (Asturias & Timmis, 1993
); homoprotocatechuate,
max 380 nm,
36 mM1 cm1 (Miller & Lipscomb, 1996
). 2,3-Dihydroxybiphenyl was used as a model substrate to assay activity against bicyclic compounds because of experience in its use and ease of handling. Activity against 1,2-dihydroxynaphthalene was determined spectrophotometrically by the method of Kuhm et al. (1991b)
. Reactions were performed in 50 mM acetic acid/NaOH buffer (pH 5·5) and the initial rate of decrease of the absorbance at 331 nm was measured. A molar absorption coefficient (
) for 1,2-dihydroxynaphthalene at 331 nm of 2·6 mM1 cm1, as calculated by Kuhm et al. (1991b)
, was used in the enzyme activity calculations. MichaelisMenten kinetics of reactions were verified by plotting reaction rates against substrate concentrations.
The range of substrate concentrations used in enzyme assays for determination of the kinetic parameters was: 1,2-dihydroxynaphthalene, 2·5500 µM; 2,3-dihydroxybiphenyl, 10 µM2 mM; 4-methylcatechol, 50 µM10 mM; homoprotocatechuate, 100 µM10 mM; catechol, 100 µM30 mM; 4-chlorocatechol, 100 µM20 mM; 3-methylcatechol, 10 µM5 mM.
The activation energy (Ea) was estimated using the Arrhenius equation for the temperature range of 3080 °C. Enzyme reactions were performed in 50 mM phosphate buffer (pH 7·5) with 200 µM 1,2-dihydroxynaphthalene. 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.
Stability analysis.
To evaluate the temperature stability of the extradiol dioxygenases, 0·5 mg ml1 of purified NahC_JF8 was incubated in 20 mM phosphate buffer (pH 7·5) at 60, 70 and 80 °C for up to 60 min. To study the influence of chelators and inhibitors on enzyme activity, 0·5 mg ml1 of purified NahC_JF8 was incubated in 20 mM phosphate buffer (pH 7·5) with 5 mM and 25 mM EDTA, and 0·1 mM and 1 mM H2O2. For NahH_JF8, cell-free extract was used as enzyme solution. After incubation at different temperatures, remaining enzyme activity was measured in 50 mM phosphate buffer (pH 7·5) with 1 mM 2,3-dihydroxybiphenyl at 25 °C.
Metal analysis.
Metal content of the purified recombinant NahC_JF8 was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Seiko SPQ6500 spectrometer. Samples for ICP-MS were prepared using acid-washed glassware. Sample and standards were prepared in 0·1 % HNO3. Separate standard curves were routinely prepared for iron and manganese and samples measured in triplicate.
RNA dot-blotting.
Bacillus sp. JF8 was grown on LB plates at 60 °C for 8 h then induced with naphthalene or biphenyl vapour by stacking the plates in a plastic container with a tight-fitting lid containing biphenyl or naphthalene crystals for 4 h. Using RNeasy Mini Kit (Qiagen), total RNA was isolated from Bacillus sp. JF8 according to the manufacturer's instructions. RNA samples (1 µg) were spotted on Hybond-N+ membranes, baked at 120 °C for 30 min, and then hybridized with DIG-labelled DNA probes which were generated by PCR using the primers derived from the two extradiol dioxygenases: nC-F (5'-ATGATTTTGCGGCTTGGT-3'), nC-R (5'-TCCAATTTTGATTGACTGGAC-3') (for nahC), and nH-F (5'-ATGTCTCACATGGGAATTTTT-3'), nH-R (5'-ACCCCTTGGCAAAAGATTCA-3') (for nahH) (Fig. 1). Detection was carried out using Anti-Digoxigenin-AP (Roche) and CSPD (Roche).
RT-PCR.
RNA samples were treated with DNase I (Invitrogen). RT-PCR was carried out with the OneStep RT-PCR kit (Qiagen) using primers nH-F (described above) and nO-R (5'-CGATGTCATTTCAATAAGCCG-3') (to amplify the region between nahH and nahO), nO-F (5'-TGAAAGTAGCGATTCTCGGA-3') and nM-R (5'-TCCCCTTCGCTGAAGATAAA-3') (nahO to nahM), nM-F (5'-CGCTTCGAGACGGTTCTCAT-3') and nC-R (described above) (nahM to nahC) (Fig. 1). The conditions for the RT-PCR amplification were as follows: 50 °C for 30 min; 95 °C for 15 min; followed by 35 cycles of 94 °C for 30 s, 48 °C for 1 min, and 72 °C for 5 min; followed by cooling to 4 °C. In the PCR amplification controls, the initial incubation at 50 °C was omitted.
Biotransformation assay.
Recombinant E. coli cells with nahC_JF8 or bphC_Q1 were cultured in LB medium with appropriate antibiotics. After 4 h of growth at 37 °C, 1 mM IPTG was added and the cells cultivated for a further 2 h. The cells were harvested, washed and suspended in 50 mM acetic acid/NaOH buffer (pH 5·5) at OD600 1. Five millilitres of the bacterial suspension was transferred to a 15 ml test tube and 1,2-dihydroxynaphthalene added at a concentration of 1 mM. The tubes were incubated at 37 °C for 2 h. The cell suspension was acidified by the addition of 50 µl concentrated HCl; an equal volume of ethyl acetate was added and mixed for 10 min. After centrifugation at 5000 g for 10 min to extract the transformants, the ethyl acetate was concentrated under a stream of nitrogen and trimethylsilylation done using BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide)+TMCS (trimethylchlorosilane) (Sylon BFT kit, Supelco). The derivatized samples were analysed by gas chromatography (Hewlett Packard model 6890), equipped with an HP-5ms capillary column (50 m, 0·2 mm, 0·33 µm, Hewlett Packard) and a mass-selective detector (Hewlett Packard, model 5972A). The conditions for GC-MS have been described previously (Shimura et al., 1999). The products of biotransformation were analysed by operating the system in the scanning mode (50700 m/z).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a phylogenetic tree of 27 extradiol dioxygenases, the enzymes cluster into three groups (Fig. 2). Ring-cleavage dioxygenases which exhibit a preference for monocyclic substrates cluster into one group (designated IB) while dioxygenases with a preference for bicyclic substrates form a second group (designated IC). The third group (designated IA) consists of the smaller extradiol dioxygenases which are single-domain enzymes, unlike the enzymes of the two previous groups, which have two identical domains. NahC_JF8 and NahH_JF8 cluster with group IB. The enzymes in this group appear to comprise three subgroups. While NahH_JF8 is in a subgroup by itself (IBa), NahC_JF8 and BphC_JF8 belong to the same subgroup (IBc). Subgroup IBc has several enzymes derived from extremophiles: HPCD_Oi (38 % identity to NahC_JF8) derived from Oceanobacillus iheyensis HTE831, an extremely halotolerant and alkaliphilic deep-sea bacterium (Lu et al., 2001
); HPCD_R1 (35 % identity), from Deinococcus radiodurans, a radioresistant bacterium (White et al., 1999
); C23O_Ss (31 % identity), from Sulfolobus solfataricus P2, an extremely thermoacidophilic archaeon (She et al., 2001
); and PheB_FDTP3 (28 % identity) from the thermophilic Bacillus stearothermophilus FDTP-3 (Dong et al., 1992
). MndD_CM2 (33 % identity), a Mn(II)-dependent HPCD which exhibits high stability to metal chelators (Whiting et al., 1996
), and HPCD_Bf (38 % identity), a stable Fe(II)-dependent HPCD which exhibits catalase and dioxygenase activity (Miller & Lipscomb, 1996
), are also in this subgroup.
|
|
|
Biochemical characterization of NahC_JF8 and NahH_JF8
Cell-free extracts from the recombinant E. coli cells expressing NahC_JF8 exhibited a specific activity of 0·06 U mg1 with 2,3-dihydroxybiphenyl. NahC_JF8 could be purified 25-fold with an overall yield of 70 % using a HiTrap Q ion-exchange column at room temperature, after thermal denaturation of the mesophilic proteins in the cell extract by treatment at 60 °C for 100 min (see Methods) (Table 2). In the case of recombinant NahH_JF8, the active enzyme could not be purified, although the heat treatment was omitted and the cell extract was directly loaded on to a HiTrap Q ion-exchange column at 4 °C. The addition of solvents such as ethanol, 2-propanol and acetone did not increase the stability of NahH_JF8, nor did addition of reducing agents such as
-mercaptoethanol and ascorbate, or changing the composition of phosphate buffer (20 mM to 100 mM). NahH_JF8 in cell-free extract retained more than 95 % activity when incubated for 9 h at 4 °C. Therefore, the biochemical characterization of NahH_JF8 was done using cell-free extracts of recombinant E. coli.
|
The Km value for 1,2-dihydroxynaphthalene of NahC_JF8 is 16 times lower than that for 2,3-dihydroxybiphenyl and 62 times lower than that for 4-methylcatechol (Table 3). The substrate preference of NahC_JF8 as based on Km value was in the order 1,2-dihydroxynaphthalene>2,3-dihydroxybiphenyl>4-methylcatechol>3-methylcatechol>4-chlorocatechol>homoprotocatechuate. Thus, the substrate preference of NahC_JF8 exhibited nearly the same trend as known 1,2-dihydroxynaphthalene dioxygenases (Patel & Barnsley, 1980
; Hirose et al., 1994
; Kuhm et al., 1991b
). An increase in the temperature (from 25 °C to 60 °C) caused a decrease in the Km exhibited by NahC_JF8, while a substantial increase in the Vmax ensured that the specificity constants (kcat/Km) were higher at 60 °C (Table 3
). The optimum temperature for NahC_JF8 under our assay conditions was determined to be 80 °C and the activation energy for the meta-cleavage of 1,2-dihydroxynaphthalene by NahC_JF8 was 11·6±0·2 kcal mol1(48·5±0·8 kJ mol1). NahH_JF8, in cell-free extract, exhibited a broad substrate preference (Table 4
), with the Km ranging from 0·25 µM to 11 µM for 1,2-dihydroxynaphthalene, 2,3-dihydroxybiphenyl, catechol, 4-chlorocatechol, and 3- and 4-methylcatechol at 25 °C. NahH_JF8 exhibited activity against 1,2-dihydroxynaphthalene with a Km about 44-fold higher than that for 2,3-dihydroxybiphenyl.
|
|
|
Gene expression
To determine whether both naphthalene and biphenyl can induce NahC_JF8 and NahH_JF8, Bacillus sp. JF8 was grown on LB plates for 8 h and then incubated with naphthalene and biphenyl. Northern hybridization was done with probes for nahC_JF8 and nahH_JF8. As shown in Fig. 5(a), neither of the genes is constitutively expressed, while both genes are induced by naphthalene and not by biphenyl. RT-PCR indicated that nahHLOMmocBnahC is transcribed as a single unit (Fig. 1
, Fig. 5b
). As the genes appear to form an operon and are induced by naphthalene, they have been designated nah.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
On a phylogenetic tree, NahC_JF8, NahH_JF8 and BphC_JF8 (Hatta et al., 2003) cluster with extradiol dioxygenases which exhibit a preference for monocyclic substrates (Fig. 2
). This group of enzymes consists of three subgroups which are stable as evidenced by the bootstrap analysis, with NahH_JF8 as the sole member of subgroup IBa, and NahC_JF8 and BphC_JF8 as the members of subgroup IBc. When the relationship of the C-terminal domains, where the conserved active-site residues and metal ligands are located, was analysed, NahH_JF8 clustered with NahC_JF8 and BphC_JF8 (results not shown), indicating that the differences are mainly confined to the N-terminal domain. As the function of the N-terminal domain and the advantages it might confer on the extradiol dioxygenases are not clear, the implication of this observation is not evident.
Interestingly, of the ten extradiol dioxygenases in subgroup IBc, six (including NahC_JF8 and BphC_JF8) are encoded by extremophiles while two more (MndD_CM2 and HPCD_Bf) are exceptionally stable extradiol dioxygenases (Boldt et al., 1995; Whiting et al., 1996
; Miller & Lipscomb, 1996
).
While analysing the evolutionary relationship between extradiol dioxygenases, Eltis & Bolin (1996) evaluated the functional significance of several conserved residues. They identified two residues Asn-243 and Asp-244 (numbering as in BphC_KKS102) in enzymes which preferentially cleave bicyclic substrates. Although NahC_JF8 preferentially cleaves a hydroxylated naphthalene ring, the Asn-Asp sequence is replaced by Leu-Ser. Similarly, in BphC_JF8, for which the preferred substrate is 2,3-dihydroxybiphenyl, the Asn-Asp residue is replaced with Ile-Ser (Hatta et al., 2003
). Analysis of the amino acid sequence in the PROSITE extradiol dioxygenase fingerprint region showed that in NahH_JF8, the consensus [LIVMF], which represents residue types found at a position, is substituted by a Thr residue. As the adjacent Tyr (Y) is an active-site residue (Eltis & Bolin, 1996
), the substitution of a hydrophobic residue (LIVMF) by a hydrophilic polar residue (T) might have had an effect on the enzyme activity. However, NahH_JF8 exhibited meta-cleavage activity against a wide range of substrates, and we propose that the consensus pattern be expanded to include the presence of a Thr residue as [LIVMFT].
Native nondenaturing PAGE of the purified recombinant NahC_JF8 and recombinant NahH_JF8 in cell-free extract appeared to indicate a tetrameric and dimeric structure, respectively, for the enzymes. BphC_LB400 and BphC_KKS102 were shown to be octamers while MPC_mt2 is a tetramer (Eltis et al., 1993; Senda et al., 1996
; Kita et al., 1999
). Kita et al. (1999)
had opined that the long protruding loop at the end region of the N-terminal domain (Gly-130 to Trp-139) prevents the formation of an octameric structure in MPC_mt2. In the alignment of Fig. 3
, a similar loop can be observed in NahC_JF8, which could explain its tetrameric structure as deduced from the results of native nondenaturing PAGE.
To produce active recombinant NahC_JF8, the presence of Mn(II) ions in the medium was essential, indicating that the enzyme was a Mn(II)-dependent extradiol dioxygenase. Fe(II) was necessary for the production of active recombinant NahH_JF8, indicating that like most extradiol dioxygenases isolated so far, NahH_JF8 utilizes Fe(II). MndD_CM2 and BphC_JF8 are two Mn(II)-dependent extradiol dioxygenases which have been studied in detail (Boldt et al., 1995, 1997
; Whiting et al., 1996
; Hatta et al., 2003
). They are distinct from Fe(II)-dependent extradiol dioxygenases like BphCII_P6 and BphC_LB400, which are completely inactivated by 0·1 mM H2O2 (Asturias et al., 1994
). The presence of Mn(II) in NahC_JF8 was deduced by its stability towards H2O2 and at 70 °C; the addition of 1 mM MnCl2 prevented inactivation of NahC_JF8, indicating that the inactivation of the enzyme at 70 °C was most likely due to the loss of the metal cofactor. ICP-MS confirmed the presence of Mn(II) in NahC_JF8, although the result obtained of 1·45±0·015 g atom Mn per homotetramer indicates that it has less than stoichiometric metal content. It is possible that a partial loss of the metal cofactor occurred during the purification process.
The Km of NahC_JF8 indicates its affinity for 1,2-dihydroxynaphthalene. An increase in temperature caused a decrease in the affinity of NahC_JF8 for its substrate; however, a significant increase in the Vmax and kcat compensated for the increase in Km, actually increasing the catalytic efficiency of NahC_JF8 twofold at 60 °C. Observations of Vmax increases keeping the kcat/Km value of thermophilic enzymes in a similar range at higher temperatures have been reported in other thermophiles (Vieille et al., 1995). NahH_JF8, on the other hand, catalysed a broad range of substrates.
The thermostability of NahC_JF8 was comparable with that of BphC_JF8. Both enzymes are not inactivated at 60 °C and retain 50 % activity after 20 min incubation at 80 °C. In contrast, NahH_JF8, in cell-free extract, lost 65 % activity after 20 min at 60 °C. Although the thermostability of NahH_JF8 appears low in comparison with the two other extradiol dioxygenases from Bacillus sp. JF8, its thermostablity is better than that of purified Fe(II)-dependent C23O from the thermophilic Bacillus thermoleovorans A2 (50 % loss of activity at 57 °C in 4·8 min and at 62 °C in 3·3 min; Milo et al., 1999) and mesophilic extradiol dioxygenases like BphC of P. putida OU83 (complete loss of activity in 10 min at 65 °C; Khan et al., 1996
) and BphCII from Rhodococcus globerulus P6 (90 % loss of activity at 50 °C in 10 min; Asturias et al., 1994
). We were unable to purify recombinant NahH_JF8. It has been reported that C23Os are easily inactivated by various oxidizing regents, such as air or H2O2 (Nozaki et al., 1968
) and the inactivation is due to oxidation of the active-site Fe(II) to Fe(III). Purification of NahH_JF8 under anaerobic conditions might yield active NahH_JF8.
Although the in vivo role of the two extradiol dioxygenases could not be confirmed, as we were unable to transform Bacillus sp. JF8, Northern hybridization indicated that both NahC_JF8 and NahH_JF8 are induced in the presence of naphthalene and not biphenyl. RT-PCR studies showed that nahHLOMmocBnahC is transcribed as a single operon. In all nah or nah-like genes (pah, nag, nid) studied so far, the extradiol dioxygenase genes have been found coupled with the genes encoding the ring-hydroxylating dioxygenase, the dihydrodiol dehydrogenase and the hydrolytic enzyme which acts on the ring-cleavage product (Simon et al., 1993; Bosch et al., 1999
; Kiyohara et al., 1994
; Fuenmayor et al., 1998
; Treadway et al., 1999
). However, in JF8, the above-mentioned upper pathway genes were not found in association with nahC, although it is possible that some of the genes found in the operon could have activities similar to upper operon genes. Instead the operon was flanked by ORFs whose predicted amino acid sequences exhibited homology to transposases (Fig. 1
). The presence of genes involved in the degradation of aromatic compounds on transposable elements has been noted before (Tsuda & Genka, 2001
; Wyndham et al., 1994a
) and the role of transposable elements in the horizontal transfer of genes is well established (Herrick et al., 1997
; Wyndham et al., 1994b
). Recruitment of catabolic transposable elements by bacteria would enhance the ability of the micro-organism to occupy new ecological niches. However, acquiring only a part of a metabolic pathway would not be useful, therefore the other genes and proteins which play a role in naphthalene metabolism must have been acquired separately and the chromosomally encoded upper pathway genes specific for naphthalene are present elsewhere in Bacillus sp. JF8.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asturias, J. A., Eltis, L. D., Prucha, M. & Timmis, K. N. (1994). Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. Identification of a new family of extradiol dioxygenases. J Biol Chem 269, 78077815.
Axcell, B. C. & Geary, P. J. (1975). Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. Biochem J 146, 173183.[Medline]
Bayly, R. C., Dagley, S. & Gibson, D. T. (1966). The metabolism of cresols by species of Pseudomonas. Biochem J 101, 293301.[Medline]
Boldt, Y. R., Sadowsky, M. J., Ellis, L. B., Que, L., Jr & Wackett, L. P. (1995). A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family. J Bacteriol 177, 12251232.[Abstract]
Boldt, Y. R., Whiting, A. K., Wagner, M. I., Sadowsky, M. J., Que, L., Jr & Wackett, L. P. (1997). Manganese(II) active site mutants of 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis strain CM-2. Biochemistry 36, 21472153.[CrossRef][Medline]
Bosch, R., Garcia-Valdes, E. & Moore, E. R. (1999). Genetic characterization and evolutionary implications of a chromosomally encoded naphthalene-degradation upper pathway from Pseudomonas stutzeri AN10. Gene 236, 149157.[CrossRef][Medline]
Buswell, J. A. (1974). The meta-cleavage of catechol by a thermophilic Bacillus species. Biochem Biophys Res Commun 60, 934941.[Medline]
Buswell, J. A. (1975). Metabolism of phenol and cresols by Bacillus stearothermophilus. J Bacteriol 124, 10771083.[Medline]
Chen, C. & Taylor, R. (1995). Thermophilic biodegradation of BTEX by two Thermus species. Biotechnol Bioeng 48, 614624.
Dong, F. M., Wang, L. L., Wang, C. M., Cheng, J. P., He, Z. Q., Sheng, Z. J. & Shen, R. Q. (1992). Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli. Appl Environ Microbiol 58, 25312535.[Abstract]
Duffner, F. M. & Muller, R. (1998). A novel phenol hydroxylase and catechol 2,3-dioxygenase from the thermophilic Bacillus thermoleovorans strain A2: nucleotide sequence and analysis of the genes. FEMS Microbiol Lett 161, 3745.[CrossRef][Medline]
Duffner, F. M., Kirchner, U., Bauer, M. P. & Muller, R. (2000). Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway. Gene 256, 215221.[CrossRef][Medline]
Eaton, R. W. & Chapman, P. J. (1992). Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions. J Bacteriol 174, 75427554.[Abstract]
Eltis, L. D. & Bolin, J. T. (1996). Evolutionary relationships among extradiol dioxygenases. J Bacteriol 178, 59305937.[Abstract]
Eltis, L. D., Hofmann, B., Hecht, H. J., Lunsdorf, H. & Timmis, K. N. (1993). Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem 268, 27272732.
Ensley, B. D., Gibson, D. T. & Laborde, A. L. (1982). Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816. J Bacteriol 149, 948954.[Medline]
Fuenmayor, S. L., Wild, M., Boyes, A. L. & Williams, P. A. (1998). A gene cluster encoding steps in conversion of naphthalene to gentisate in Pseudomonas sp. strain U2. J Bacteriol 180, 25222530.
Haddock, J. D., Nadim, L. M. & Gibson, D. T. (1993). Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400. J Bacteriol 175, 395400.[Abstract]
Han, S., Eltis, L. D., Timmis, K. N., Muchmore, S. W. & Bolin, J. T. (1995). Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad. Science 270, 976980.[Abstract]
Hatta, T., Mukerjee-Dhar, G., Damborsky, J., Kiyohara, H. & Kimbara, K. (2003). Characterization a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB and naphthalene-degrading Bacillus sp. JF8. J Biol Chem 278, 2148321492.
Herrick, J. B., Stuart-Keil, K. G., Ghiorse, W. C. & Madsen, E. L. (1997). Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site. Appl Environ Microbiol 63, 23302337.[Abstract]
Hirose, J., Kimura, N., Suyama, A., Kobayashi, A., Hayashida, S. & Furukawa, K. (1994). Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin. FEMS Microbiol Lett 118, 273277.[CrossRef][Medline]
Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J. (1990). PCR Protocols: a Guide to Methods and Applications. San Diego, California: Academic Press.
Khan, A. A., Wang, R. F., Nawaz, M. S., Cao, W. W. & Cerniglia, C. E. (1996). Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC). Appl Environ Microbiol 62, 18251830.[Abstract]
Kita, A., Kita, S., Fujisawa, I., Inaka, K., Ishida, T., Horiike, K., Nozaki, M. & Miki, K. (1999). An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2. Structure Fold Des 7, 2534.[Medline]
Kiyohara, H., Torigoe, S., Kaida, N., Asaki, T., Iida, T., Hayashi, H. & Takizawa, N. (1994). Cloning and characterization of a chromosomal gene cluster, pah, that encodes the upper pathway for phenanthrene and naphthalene utilization by Pseudomonas putida OUS82. J Bacteriol 176, 24392443.[Abstract]
Kuhm, A. E., Stolz, A. & Knackmuss, H. J. (1991a). Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1. Biodegradation 2, 115120.[Medline]
Kuhm, A. E., Stolz, A., Ngai, K. L. & Knackmuss, H. J. (1991b). Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids. J Bacteriol 173, 37953802.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lu, J., Nogi, Y. & Takami, H. (2001). Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol Lett 205, 291297.[CrossRef][Medline]
Miller, M. A. & Lipscomb, J. D. (1996). Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum. A dioxygenase with catalase activity. J Biol Chem 271, 55245535.
Milo, R. E., Duffner, F. M. & Muller, R. (1999). Catechol 2,3-dioxygenase from the thermophilic, phenol-degrading Bacillus thermoleovorans strain A2 has unexpected low thermal stability. Extremophiles 3, 185190.[CrossRef][Medline]
Nozaki, M., Ono, K., Nakazawa, T., Kotani, S. & Hayaishi, O. (1968). Metapyrocatechase. II. The role of iron and sulfhydryl groups. J Biol Chem 243, 26822690.
Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357358.[Medline]
Patel, T. R. & Barnsley, E. A. (1980). Naphthalene metabolism by pseudomonads: purification and properties of 1,2-dihydroxynaphthalene oxygenase. J Bacteriol 143, 668673.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Senda, T., Sugiyama, K., Narita, H., Yamamoto, T., Kimbara, K., Fukuda, M., Sato, M., Yano, K. & Mitsui, Y. (1996). Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102. J Mol Biol 255, 735752.[CrossRef][Medline]
She, Q., Singh, R. K., Confalonieri, F. & 28 other authors (2001). The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci U S A 98, 78357840.
Shimura, M., Mukerjee-Dhar, G., Kimbara, K., Nagato, H., Kiyohara, H. & Hatta, T. (1999). Isolation and characterization of a thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene. FEMS Microbiol Lett 178, 8793.[CrossRef][Medline]
Simon, M. J., Osslund, T. D., Saunders, R. & 7 other authors (1993). Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene 127, 3137.[CrossRef][Medline]
Sterner, R. & Liebl, W. (2001). Thermophilic adaptation of proteins. Crit Rev Biochem Mol Biol 36, 39106.
Taira, K., Hayase, N., Arimura, N., Yamashita, S., Miyazaki, T. & Furukawa, K. (1988). Cloning and nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene from the PCB-degrading strain of Pseudomonas paucimobilis Q1. Biochemistry 27, 39903996.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Treadway, S. L., Yanagimachi, K. S., Lankenau, E., Lessard, P. A., Stephanopoulos, G. & Sinskey, A. J. (1999). Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24. Appl Microbiol Biotechnol 51, 786793.[CrossRef][Medline]
Tsuda, M. & Genka, H. (2001). Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53. J Bacteriol 183, 62156224.
Vieille, C., Hess, J. M., Kelly, R. M. & Zeikus, J. G. (1995). xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol 61, 18671875.[Abstract]
Vieille, C., Burdette, D. S. & Zeikus, J. G. (1996). Thermozymes. Biotechnol Annu Rev 2, 183.[Medline]
White, O., Eisen, J. A., Heidelberg, J. F. & 23 other authors (1999). Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286, 15711577.
Whiting, A. K., Boldt, Y. R., Hendrich, M. P., Wackett, L. P. & Que, L., Jr (1996). Manganese(II)-dependent extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis CM-2. Biochemistry 35, 160170.[CrossRef][Medline]
Wyndham, R. C., Cashore, A. E., Nakatsu, C. H. & Peel, M. C. (1994a). Catabolic transposons. Biodegradation 5, 323342.[Medline]
Wyndham, R. C., Nakatsu, C., Peel, M., Cashore, A., Ng, J. & Szilagyi, F. (1994b). Distribution of the catabolic transposon Tn5271 in a groundwater bioremediation system. Appl Environ Microbiol 60, 8693.[Abstract]
Yeh, W. K., Gibson, D. T. & Liu, T. N. (1977). Toluene dioxygenase: a multicomponent enzyme system. Biochem Biophys Res Commun 78, 401410.[Medline]
Received 22 October 2003;
revised 22 December 2003;
accepted 2 January 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |