Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: Gene sequence and enzyme characterization

Article abstract of DOI:10.1271/bbb.68.1798

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.

Full text of DOI:10.1271/bbb.68.1798

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Catechol 2,3-Dioxygenase from Pseudomonas
sp. Strain ND6: Gene Sequence and Enzyme
Characterization
Yun JIANGa, Xianglong YANGb, Bin LIUc, Huabing ZHAOa, Qiuxiang CHENGa & Baoli CAIa
a Department of Microbiology, Nankai University
b Department of Laboratory Science, Tianjin Medical University
c Genomics Institute, Chinese Academy of Sciences
Published online: 22 May 2014.
To cite this article: Yun JIANG, Xianglong YANG, Bin LIU, Huabing ZHAO, Qiuxiang CHENG & Baoli CAI (2004) Catechol 2,3-
Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization, Bioscience, Biotechnology,
and Biochemistry, 68:8, 1798-1800, DOI: 10.1271/bbb.68.1798
To link to this article: http://dx.doi.org/10.1271/bbb.68.1798
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Biosci. Biotechnol. Biochem., 68 (8), 1798–1800, 2004
Note
Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6:
Gene Sequence and Enzyme Characterization
y
1;
Yun JIANG,1 Xianglong YANG,2 Bin LIU,3 Huabing ZHAO,1 Qiuxiang CHENG,1 and Baoli CAI
1Department of Microbiology, Nankai University, Tianjin 300071, China
2Department of Laboratory Science, Tianjin Medical University, Tianjin 300020, China
3Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China
Received April 14, 2004; Accepted May 27, 2004
The catechol 2,3-dioxygenase (C23O) gene in naph-
thalene catabolic plasmid pND6-1 of Pseudomonas sp.
ND6 was cloned and sequenced. The C23O gene was
consisted of 924 nucleotides and encoded a polypeptide
of molecular weight 36 kDa containing 307 amino acid
residues. The C23O of Pseudomonas sp. ND6 exhibited
93% and 89% identities in amino acid sequence with
C23Os encoded by naphthalene catabolic plasmid
NAH7 from Pseudomonas putida G7 and the chromo-
some of Pseudomonas stutzeri AN10 respectively. The
Pseudomonas sp. ND6 C23O gene was overexpressed in
Escherichia coli DH 5ꢀ using the lac promoter of
pUC18, and its gene product was purified by DEAE-
Sephacel and Phenyl-Sepharose CL-4B chromatogra-
phy. The enzymology experiments indicated that the
specific activity and thermostability of C23O from
Pseudomonas sp. ND6 were better than those of C23O
from Pseudomonas putida G7.
breaking the C–C bond at the 2,3-(meta) position, which
is part of the ꢁ-ketoadipate pathway.2) Many research
works have been devoted to different aspects of C23O
for its potential theoretical and applied values in
environmental protection and other areas.3) At present,
over 30 extradiol catechol dioxygenases has been
reported4) and the nucleotide sequences of some cat-
echol 2,3-dioxygenase genes has been determined.2,5–8)
Previously we isolated a naphthalene-degrading bac-
terium, strain ND6. The naphthalene catabolic genes of
this strain were localized on the 102 kb plasmid, pND6-
1, in a DNA hybridization experiment.9) In this study,
we determined and analyzed the 16S rRNA gene
sequence of strain ND6, cloned, expressed, and se-
quenced the C23O gene of strain ND6, and purified and
characterized the catechol 2,3-dioxygenase expressed.
The primer pair 27F (AGAGTTTGATCMTGGCT-
CAG) and 1492R (CGGYTACCTTGTTACGACTT)10)
was used for PCR amplification of 16S rRNA gene. The
PCR product was cloned into the pMD 18-T vector
(Takara Biotechnology Co.) following the manufactur-
er’s instructions. Primers targeted at the T7 and Sp6
promoters and a universal primer 533F (GTGCCA-
GCMGCCGCGGTAA)10) were used for sequencing to
cover the entire length of the 16S rRNA gene. The final
sequence of 1499 bp was submitted to the NCBI
GenBank under accession no. AY589689. The BLAST
search of this sequence indicated that it was matched at
99% to 33 strains of Pseudomonas. Hence strain ND6
was identified as a Pseudomonas sp.
Key words: Pseudomonas sp. ND6; catechol 2,3-dioxy-
genase; gene sequence; enzyme character-
ization
Many microorganisms are able to utilize xenobiotic
aromatic compounds as the sole carbon and energe
source for growth. Various efforts have been made to
improve their capabilities for environmental pollutant
degradation and bioremediation.1) The aromatic com-
pounds, including biphenyl, naphthalene, toluene, phe-
nol, and benzoates can be converged to catechol as a
catabolic intermediate by microbial degradation. The
catechol formed as an intermediate is subjected to its
ring fission by catechol 1,2-dioxygenase or catechol 2,3-
dioxygenase. The catechol 1,2-dioxygenase is an intra-
diol-type enzyme which cleaves the C–C bond at the
1,2-(ortho) position of catechol to make cis,cis-muconic
acid, which is further degraded by the ꢀ-ketoadipate
pathway. The catechol 2,3-dioxygenase (C23O), an
extradiol-ring fission enzyme, catalyzes the conversion
of catechol to 2-hydroxymuconic semialdehyde by
Plasmid DNA of Pseudomonas sp. ND6 was isolated
by the SDS-alkali lysis method11) and purified by CsCl-
ethidium bromide ultracentrifugation.12) To construct a
plasmid shotgun library, the purified plasmid DNA was
broken by sonication and DNA fragments of 1ꢀ3 kb in
size were isolated by agarose gel electrophoresis. The
DNA ends were filled in, ligated into plasmid pUC18,
and transformed into E. coli DH 5ꢁ by electropora-
tion.12) White clones containing inserts were randomly
selected. DNA templates from white clones were
y
To whom correspondence should be addressed. Tel: +86-22-23503617; Fax: +86-22-23508800; E-mail: caibaoli@nankai.edu.cn
Abbreviations: C23O, catechol 2,3-dioxygenase; IPTG, isopropylthio-ꢀ-D-galactoside; PAGE, polyacrylamide gel electrophoresis

Characterization of Catechol 2,3-Dioxygenase
1799
prepared from 1 ml overnight cultures using the alkaline
lysis method.12) Each of cloned fragments was se-
quenced from both ends by using an ABI model 377A
DNA sequencer (Applied Biosystems, Foster city,
California, USA). The DNA sequences in the plasmid
shotgun library were submitted to a BLAST search of
the NCBI GenBank database to identify naphthalene
degradation genes. Computational analysis indicated
that 23 genes were predicted to be involved in
naphthalene catabolism. The complete nucleotide se-
quence of pND6-1 from strain ND6 has been deposited
in GenBank under accession no. AY208917 (Wei Li and
Baoli Cai, unpublished).
In the sequence library of pND6-1, we found that the
cloning fragment of 1551 bp (no. 0842) included a
coding sequence of C23O. The recombinant plasmid
containing the C23O gene was designated pNDN. The
C23O gene was consisted of 924 nucleotides and
encoded a polypeptide of molecular weight 36 kDa
containing 307 amino acid residues. The sequence of
catechol 2,3-dioxygenase of Pseudomonas sp. ND6
exhibited 95% homology at the nucleotide level and
93% homology at the amino acid level with the
corresponding enzyme from Pseudomonas putida G75)
(Fig. 1). Ishida et al. suggested that C23O from
Pseudomonas putida mt-2 has five conserved active-site
residues, His153, His199, His214, Tyr255, and
Glu265.4) These conserved active-site residues were
not changed in the C23O of strain ND6. This study
indicated that globally distributed catechol 2,3-dioxyge-
nase genes are highly conserved among different
bacterial species.
recombinant plasmid, pNDG, containing the C23O gene
from Pseudomonas putida G7 using pUC18 as a
vector.9) E. coli DH 5ꢁ containing pNDN or pNDG
were grown in LB medium supplemented with ampi-
cillin for 12 h, then IPTG was added at a final
concentration of 0.1 mM and growth continued for 4 h
to induce gene expression. The cells were harvested by
centrifuging 100 ml of culture and washed with 20 mM
sodium phosphate buffer (pH7.5). Pelleted cells were
resuspended in 8 ml of the same buffer, 2 ml of cold
acetone was added, and then the mixture was subjected
to sonication with a cell disruptor. The crude extract was
obtained by centrifugation at 13;682 ꢁ g for 30 min at
4 ꢂC. The supernatant was fractionated by 20%, 40%,
and 60% (NH4)2SO4 saturations. The precipitates of the
40% and 60% (NH4)2SO4 saturations were dissolved in
2 ml of 20 mM sodium phosphate buffer, mixed, and
dialyzed overnight against the same buffer. The dialy-
sate was applied to a DEAE-Sephacel column (2 ꢁ 30
cm) preequilibrated with 20 mM sodium phosphate
buffer. Proteins in the column were eluted with a linear
NaCl gradient up to 0.7 M in 20 mM sodium phosphate
buffer. Fractions with C23O activity were pooled and
then applied to a Phenyl-Sepharose CL-4B column
(1:5 ꢁ 20 cm) preequilibrated with 50 mM sodium phos-
phate buffer (pH7.5). Proteins were eluted with a linear
gradient of 50 mM sodium phosphate buffer and distilled
water. Fractions with C23O activity were pooled and
concentrated by ultrafiltration. C23O activity was
measured spectrophotometrically following the forma-
tion of 2-hydroxymuconic semialdehyde from catechol
at 375 nm. Activity assays were performed in 50 mM
sodium phosphate buffer containing 0.5 mM catechol as
substrate. One unit of enzyme activity was defined as the
With the exception of pNDN containing the Pseudo-
monas sp. ND6 C23O gene, we also constructed a
Fig. 1. Comparison of Deduced Amino Acid Sequences of Catechol 2,3-Dioxygenases from Pseudomonas sp. Strain ND6 and Pseudomonas
putida G7.
The different amino acid residues of the two enzymes are indicated by frames.

1800
Y. JIANG et al.
ND6 were better than those of C23O from Pseudomonas
putida G7.
1
2
3
4
5
Acknowledgments
kD
97
66
43
This work was supported by grant no. 30270274 from
the National Natural Science Foundation of China.
References
1) Dua, M., Singh, A., Sethunathan, N., and Johri, A. K.,
Biotechnology and bioremediation: successes and limi-
tations. Appl. Microbiol. Biotechnol., 59, 143–152
(2002).
31
2) Moon, J., Chang, H., Min, K. R., and Kim, Y., Cloning
and sequencing of the catechol 2,3-dioxygenase gene of
Alcaligenes sp. KF711. Biochem. Biophys. Res. Com-
mun., 208, 943–949 (1995).
20
14
3) Jiang, T., Ji, C. N., Sheng, X. Y., Chen, M. Q., Xie, Y.,
Gong, W. M., and Mao, Y. M., Crystal structure of
thermostable catechol 2,3-dioxygenase determined by
multiwavelength anomalous dispersion method. Chinese
Science Bulletin, 47, 307–309 (2002).
4) Ishida, T., Kita, A., Miki, K., Nozaki, M., and Horiike,
K., Structure and reaction mechanism of catechol 2,3-
dioxygenase (metapyrocatechase). International Con-
gress Series, 1233, 213–220 (2002).
5) Ghosal, D., You, I. S., and Gunsalus, I. C., Nucleotide
sequence and expression of gene nahH of plasmid NAH7
and homology with gene xylE of TOL plasmid pWWO.
Gene, 55, 19–28 (1987).
6) Bartilson, M., and Shingler, V., Nucleotide sequence and
expression of the catechol 2,3-dioxygenase gene of
phenol-degrading Pseudomonas CF600. Gene, 85, 233–
238 (1989).
Fig. 2. SDS-PAGE of Catechol 2,3-Dioxygenases from Pseudomo-
nas sp. Strain ND6.
(1) crude cell extract; (2) 40% and 60% ammonium sulfate
fractions; (3) partially purified enzyme by DEAE- Sephacel
chromatography; (4) purified enzyme by DEAE-Sephacel chroma-
tography and Phenyl-Sepharose CL-4B chromatography; (5) Protein
molecular weight markers.
amount of enzyme that converts 1 ꢂmol of substrate per
minute. The specific activity of the enzyme was defined
in terms of units per mg of protein.2) Enzyme kinetic
parameters were determined by double-reciprocal plots.
The thermostability of C23O was determined by
incubating an enzyme solution at 57 ꢂC in 50 mM
sodium phosphate buffer. C23O activity was determined
every 5 min.
7) He, Z. Q., Mao, Y. M., Sheng, Z. J., and Shen, R. Q.,
Complete nucleotide sequence of pheB gene encoding
catechol 2,3-dioxygenase from Bacillus stearothermo-
philus. Chin. Biochem., 11, 114–116 (1995).
8) Oh, J. M., Kang, E., Min, K. R., Kim, C.-K., Kim, Y.-C.,
Lim, J.-Y., Lee, K.-S., Min, K.-H., and Kim, Y.,
Structure of catechol 2,3-dioxygenase gene encoded in
TOM plasmid of Pseudomonas cepacia G4. Biochem.
Biophys. Res. Commun., 234, 578–581 (1997).
9) Zhang, X., Yue, X., Huang, J., Niu, S., and Cai, B.,
Isolation and identification of naphthalene-degrading
plasmid pND6. Chin. J. Appl. Environ. Biol., 6, 187–190
(2000).
10) Lane, D. J., 16S/23S rRNA sequencing. In ‘‘Nucleic
Acid Techniques in Bacterial Systematics’’, eds. Stack-
ebrandt, E., and Goodfellow, M., John Wiley & Sons,
Chester, UK, pp. 371–375 (1991).
11) Cai, B., Gao, C., and Jiao, R., A simple method for the
isolation of large plasmids in Pseudomonas. Acta
Scientiae Circumstantiae, 4, 291–295 (1984).
Overexpression of the C23O gene of Pseudomonas
sp. ND6 in E. coli DH 5ꢁ was demonstrated by
determining the C23O activity and SDS-PAGE of the
crude extract (Fig. 2). The purified C23O exhibited a
single band of about 36 kDa on SDS-PAGE (Fig. 2).
The enzyme kinetic experiments indicated that Km and
the specific activity of the C23O of strain ND6 for
catechol were 11 ꢂM and 11.96 U/mg of protein re-
spectively, higher than those of the C23O from
Pseudomonas putida G7 (4.5 ꢂM and 7.43 U/mg of
protein respectively). The thermostability of the C23O
from strain ND6 was better than that of the C23O from
Pseudomonas putida G7. After incubating at 57 ꢂC for
10 min, the residual activity of ND6 C23O was 37%,
while that of G7 C23O was 28% under the same
conditions.
In conclusion, we have cloned and expressed a
catechol 2,3-dioxygenase gene from naphthalene-de-
grading Pseudomonas sp. strain ND6. The gene product
was purified by DEAE-Sephacel and Phenyl-Sepharose
CL-4B chromatography. The specific activity and
thermostability of the C23O from Pseudomonas sp.
12) Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular
cloning: a laboratory manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York
(1989).