Gentisate 1,2-Dioxygenase from Xanthobacter polyaromaticivorans 127W
193
to that of the extradiol dioxygenase group, and that the
active center contains a 2-His-1-carboxylate facial triad
that coordinates ferrous ion.6) However, phylogenetic
analyses indicate that these three groups of ring fission
dioxygenases form structurally different clusters and
originate from different ancestors.1)
GGGTCGACTTTCAC-30) primers, which were de-
signed according to the gene sequences (accession no.,
AB121977). Another forward primer (50-CTATACA-
GCGCTCCATATGGTGCTAAA-30) was used in the
production of dGDOxp with N-terminal peptide trunca-
tion from S2 to W29. Underlines represent NdeI and SalI
restriction sites respectively, and an initiation ATG
codon and a sequence complementary to the termination
TGA codon are shown in italics. PCR was performed in
30 cycles of 95 ꢀC for 1 min, 58 ꢀC for 1 min, and 74 ꢀC
for 1 min, using KOD plus DNA polymerase (Toyobo,
Osaka, Japan) and a thermal cycler GeneAmp PCR
system 2400 (PerkinElmer, Foster city, CA). Each DNA
fragment was digested with NdeI and SalI and ligated
into the NdeI-SalI gap of plasmid pET25b to create
gene expression plasmid pET25b-GDOxp or pET25b-
dGDOxp. Overexpression of the gene was induced by
the addition of 0.5 mM IPTG to a mid-log phase culture
(OD660 ¼ 0:5) in L broth at 25 ꢀC for 4 h.
Xanthobacter polyaromaticivorans 127W is capable
of degrading and growing on various 2-ring and 3-ring
polycyclic aromatic hydrocarbons (PAHs) and hetero-
cyclic aromatic compounds (HACs), such as dibenzo-
thiophene.7) It has been found that the strain degraded
these aromatic compounds even under extremely low
oxygen (ELO) conditions, where the dissolved oxygen
concentration was lower than or equal to 0.2 ppm. We
have reported information about the dbd gene cluster
responsible for degradation of both PAHs and HACs
from strain 127W, which is functional under ELO
conditions.8) The gene cluster contained a gene encoding
putative gentisate 1,2-dioxygenase (dbdB), suggesting
that gentisate is an intermediate in the PAHs and HACs
degradation pathways of the strain. In this paper, we
describe the production and characterization of recombi-
nant GDO from strain 127W, GDOxp. GDOxp was
found to be highly active against both gentisate and
dihydroxynaphthoate. GDOxp had higher affinity to
oxygen than other dioxygenase enzymes. The results
also suggest that ferrous ion specifically functions in the
formation of the active tetramer structure of GDOxp.
Construction of pSTV28-GroES/EL. In order to
promote proper folding of recombinant GDOxp in
E. coli BL21(DE3), another gene expression plasmid,
pSTV28-GroES/EL, was constructed and co-expressed
in the cells. A gene locus containing sigma34-promoter,
groEL, and groES (AE000487) was amplified by PCR
with a set of primers and genomic DNA of E. coli
BL21(DE3). The sequence of primers was: forward, 50-
GTGCTGATCAGAATTCTTTTTCTTTTTCCC-30 and
reverse, 50-GTTTGTTTATGTCGACGAGGTGCAG-30.
Underlines show recognition sites EcoRI and SalI re-
spectively. The DNA fragment was digested with EcoRI
and SalI and ligated into the EcoRI-SalI gap of pSTV28.
Gene expression was induced as described above.
Materials and Methods
Cells and plasmids. X. polyaromaticivorans 127W
was isolated from bottom sludge of a crude oil reservoir
tank in Fukui, Japan, for its ability to grow on dibenzo-
thiophene (DBT) under ELO conditions.7) E. coli JM109
and BL21 (DE3) (Stratagene, Heidelberg, Germany)
were used in gene manipulation and protein production
experiments respectively. Gene expression plasmids
pET25b and pSTV28 were purchased from Novagen
(Madison, WI) and Takara Bio (Ohtsu, Japan) respec-
tively. All the recombinant cells were grown in L broth
containing 50 mg/l ampicillin, unless otherwise stated.
L broth contains 10g/l Tryptone (Difco, Sparks, MD) and
5 g/l each of Yeast extract (Difco) and NaCl (pH 7.2).
Purification of the enzyme.
Production of GDOxp. After induction of the gene for
2 h, cells were harvested by centrifugation, and sus-
pended in 10 mM MOPS buffer (pH 7.2) containing 10%
glycerol (buffer A). The cells were then disrupted by
sonication with a model 450 sonifier (Branson Ultra-
sonic, Danbury, CT) on ice, and centrifuged at 20,000 g
for 30 min to separate soluble (cell lysate) and insoluble
(membrane and protein aggregates) fractions. The
soluble fraction was used in the purification of GDOxp.
Fractionation by ammonium sulfate. The soluble
fraction was further fractionated by stepwise increase
in the concentration of ammonium sulfate. Proteins that
were soluble at 30% and insoluble at 50% saturation of
ammonium sulfate were collected by centrifugation at
20,000 g for 30 min (4 ꢀC), and dissolved in an appro-
priate volume of buffer A. The protein solution was
dialyzed against 10 mM MOPS buffer (pH 7.2) for 16 h.
After dialysis, sediments were removed by centrifuga-
tion and the supernatant was pooled.
Prediction of secondary structure. Fully automatic
Jpred WWW server (Barton Group, Dundee, UK) was
used in prediction of protein secondary structure.9)
A multiple amino acid sequence alignment of GDOxp
and other related proteins was constructed using
CLUSTAL W.10)
Construction of pET25b-GDOxp gene expression
plasmid. Genomic DNA of strain 127W was extracted
and purified as previously described.8) The dbdB gene
was amplified by PCR with a combination of synthetic
oligonucleotide primers, forward (50-GAGGGCCACA-
TATGTCATTCGCG-30) and reverse (50-CCGAAGC-
Anion-exchange chromatography. Further purifica-
tion steps were performed using a Fast Protein Liquid
Chromatography system (Amersham Biosciences, Buck-