Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W

Article abstract of DOI:10.1271/bbb.60444

A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP 766750, 64%), and P. aeruginosa (ZP 00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.

Full text of DOI:10.1271/bbb.60444

Biosci. Biotechnol. Biochem., 71 (1), 192–199, 2007
Gentisate 1,2-Dioxygenase from Xanthobacter polyaromaticivorans 127W
Shin-ichi HIRANO,1; Masaaki MORIKAWA,
Kazufumi TAKANO,1
*
1;**;y
1
Tadayuki IMANAKA,2 and Shigenori KANAYA
1Department of Material and Life Science, Graduate School of Engineering, Osaka University,
Suita, Osaka 565-0871, Japan
2Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Kyoto 615-8510, Japan
Received August 10, 2006; Accepted September 6, 2006; Online Publication, January 7, 2007
[doi:10.1271/bbb.60444]
A putative gentisate 1,2-dioxygenase was encoded in
the dibenzothiophene degradation gene cluster (dbd)
from Xanthobacter polyaromaticivorans 127W. The de-
duced amino acid sequence showed high sequence
similarity with gentisate dioxygenases from Pseudomo-
nas alcaligenes (AAD49427, 65% identical), Bradyrhi-
zobium japonicum (NP 766750, 64%), and P. aeruginosa
(ZP 00135722, 54%), and moderate similarity with 1-
hydroxy-2-naphthoate dioxygenase from Nocardioides
sp. KP7 (BAA31235, 33%) and salicylate dioxygenase
from Pseudaminobacter salicylatoxidans (AAQ91293,
33%). The enzyme, GDOxp, was heterologously pro-
duced in Escherichia coli and purified to homogeneity.
GDOxp formed a tetramer and exhibited high dioxyge-
nase activity against 1,4-dihydroxy 2-naphthoate as well
as gentisate, suggesting unusually broad substrate spec-
ificity. GDOxp easily released ferrous ion under unfav-
orable temperature and pH conditions to become an
inactive monomer protein. An inactive monomer pro-
tein can reconstitute a tetramer structure and restore
enzyme activity in a cooperative manner upon the
addition of ferrous ion. Chymotryptic digestion and
protein truncation experiments suggested that the N-
terminal region is important for the tetramerization of
GDOxp.
and large [ꢀ] and small [ꢁ] subunits of a terminal
oxygenase component (ꢀ3ꢁ3 or ꢀ3).3) These enzymes are
mainly involved in the initial oxygenation step of
aromatic substrates to dihydroxylated intermediates.
The second class is made up of oligomeric dioxygenases,
which are responsible for subsequent ring fission of
dihydroxylated intermediates. Although the aromatic
hydrocarbons are diverse in number of rings and mod-
ifications, they are degraded by way of a limited number
of dihydroxylated intermediates, such as catechol (1,2-
dihydroxylated benzene), protocatechuate (3,4-dihy-
droxybenzoate), and gentisate (2,5-dihydroxybenzoate).
Therefore, the enzymes responsible for the ring fission of
these compounds are the funnels in the biodegradation
pathways of aromatic hydrocarbons. These ring-fission
dioxygenases are further classified into three groups
based on the cleavage site of substrates. The first group
is intradiol dioxygenase, such as catechol 1,2-dioxyge-
nase and protocatechuate 3,4-dioxygenase, which con-
tain a ferric ion in the catalytic center of each subunit
and cleave the aromatic ring between two vicinal
hydroxyl groups to yield colorless cis, cis-muconic acid
adducts.4) The second group is extradiol dioxygenase,
such as catechol 2,3-dioxygenase, protocatechuate 4,5-
dioxygenase, and 2,3-dihydroxybiphenyl dioxygenase,
which contain a catalytic ferrous ion and cleave the
aromatic ring adjacent to two vicinal hydroxyl groups to
yield yellow hydroxymuconic semialdehyde adducts.5)
The third group enzyme is apparently different from the
two former groups because it degrades 1,4- or 2,5-
dihydroxylated substrates such as gentisate (2,5-dihy-
droxybenzoate), homogentisate, and hydroquinone. Gen-
tisate 1,2-dioxygenase (GDO, EC1.13.11.4) is included
in this group, which cleaves the aromatic ring between
the carboxyl and proximal hydroxyl groups in the aro-
matic ring to yield maleylpyruvate. It has been reported
that the catalytic mechanism of GDO is basically similar
Key words: gentisate 1,2-dioxygenase; Xanthobacter;
gentisate; naphthoate; ferric ion
Dioxygenases are enzymes that catalyze dioxygen
incorporation into various organic compounds. They
play key roles in the degradation pathway of mono- and
polycyclic aromatic hydrocarbons, including recalcitrant
and detrimental compounds.1–3) Aromatic ring dioxyge-
nases contain nonheme ion and are classified into two
classes. First-class enzymes are a two- or three-compo-
nent system that consists of a reductase, (a ferredoxin),
y
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2253; E-mail: morikawa@ees.hokudai.ac.jp
Present address: Central Research Institute of Electric Power Industry, Chiba 270-1194, Japan
Present address: Division of Bioscience, Faculty of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan
*
**

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-

194
S. HIRANO et al.
inghamshire, UK). A dialyzed sample was applied to a
HitrapQ column (5 ml, Amersham) equilibrated with
buffer A. GDOxp and dGDOxp were eluted in a linear
gradient from 0 to 1 M NaCl in buffer A.
Gel filtration chromatography. Active fractions eluted
from the HitrapQ column were pooled and loaded onto
a Hiload 16/60 column containing Superdex200pg
(Amersham) equilibrated with buffer A containing 100
mM NaCl.
Limited proteolysis. GDOxp was digested with chy-
motrypsin at 37 ꢀC for 0 to 10 min in 10 mM MOPS
buffer (pH 7.2) containing 2.5 mM DTT. The ratio of
chymotrypsin to GDOxp in the reaction mixture was 1 to
100 (w/w). The protein bands visualized with Coomas-
sie brilliant blue staining were transferred to a PVDF
membrane and subjected to amino acid sequence analy-
sis. The N-terminal amino acid sequence was deter-
mined with a pulsed liquid automated sequencing
system Procise 491 (PerkinElmer).
Analysis on ferrous ion content. Ferrous ion contents
of proteins were measured by the method of Zabinski
et al.4)
Results and Discussion
Primary structure analyses of gentisate 1,2-dioxyge-
nase (GDOxp) from X. polyaromaticivorans 127W
The amino acid sequence deduced from the nucleo-
tide sequence of dbdB (AB121977)8) indicated that it
encodes a gentisate 1,2-dioxygenase composed of 350
amino acid residues with a calculated molecular weight
of 39.3 kDa and a pI value of 5.4. This protein was
named GDOxp. GDOxp shared high sequence similarity
with GDO from Pseudomonas alcaligenes (AAD49427,
65% identical), P. aeruginosa (ZP00135722, 54%), and
Bradyrhizobium japonicum (NP766750, 64%), and
moderate similarity with 1-hydroxy-2-naphthoate diox-
ygenase from Nocardioides sp. KP7 (BAA31235, 33%)
and salicylate dioxygenase from Pseudaminobacter
salicylatoxidans (AAQ91293, 33%). These GDO family
proteins commonly have two sets of cupin domain, each
comprising a six-stranded beta barrel structure predicted
by the Jpred program (Fig. 1). A cupin domain typically
Enzyme assays. Gentisate 1,2-dioxygenase activity
was assayed spectrophotometrically by monitoring the
formation of maleylpyruvate (ꢂmax ¼ 330 nm) at 23 ꢀC.
The molecular extinction coefficient value of maleyl-
pyruvate at 330 nm was 10.2 mMꢁ1 cmꢁ1.6) The reaction
mixture contained 0.33 mM of gentisate and an appro-
priate amount of enzyme in 20 mM PIPES (pH 7.5)
buffer containing 20 mM Fe(NH4)2(SO4)2 and 2 mM cys-
teine otherwise denoted. The activity of GDOxp against
other substrates (0.33 mM) was assayed similarly. The
molecular extinction coefficient values of products from
5-aminosalicylate, 1-hydroxy 2-naphthoic acid, and 1,4-
dihydroxy 2-naphthoic acid were 12.4 (1,5-dicarboxy-3-
amino-2,4-pentadiene-1-one, ꢂmax ¼ 358 nm), 11.5 (4-
(2-carboxyphenyl)-2-oxobut-3-enoate, ꢂmax ¼ 300 nm),
and 9.2 mMꢁ1 cmꢁ1 (4-hydroxy-(2-carboxyphenyl)-2-
oxobut-3-enoate, ꢂmax ¼ 312 nm) respectively.6,11,12)
One unit of activity was defined as the amount of en-
zyme converting 1 mmol of substrate to a corresponding
product per min. Kinetic parameters were determined
from a Lineweaver–Burk plot of initial reaction velocity
against different concentrations of the substrate. The Km
value for oxygen was determined in the presence of
0.33 mM gentisate under various dissolved-oxygen con-
ditions. Different dissolved-oxygen conditions were
prepared by changing the incubation time in an
anaerobic chamber, as described previously.7) As the
incubation time increased, the oxygen concentration in
the solution decreased. The Hill coefficient was deter-
mined from a sigmoidal reactivation curve of monomer
GDOxp under different ferrous ion concentrations.13,14)
The protein concentration was determined by the
method of Bradford (BioRad Protein assay kit, BioRad
Laboratories, Hercules, CA), with bovine serum albumin
as a standard.
contains a set of motifs, motif 1, G(X)5HXH(X)3;4
-
E(X)6G, and motif 2, G(X)5PXG(X)2H(X)3N, and an
intermotif region of various lengths.16) Two histidines,
HXH in motif 1 and a histidine in motif 2, have been
reported to be essential for dioxygenase activity, and
all these histidines were completely conserved in
GDOxp.17) However, the glutamic acid in motif 1 and
the second glycine in motif 2 are commonly replaced in
GDO with alanine (A114) and tryptophane or phenyl-
alanine (W/F146) respectively. A highly conserved
glutamic acid (E103) in motif 1 might constitute a 2-
His-1-carboxylate facial triad that functions as a metal
ligand. Although GDO has been classified in the bicupin
family, which bears dual cupin domains, distinguished
residues in motif 3 and motif 4 of the possible C-
terminal domain were less conserved than those in motif
1 and motif 2 of the N-terminal domain.
Overproduction and purification of GDOxp
CD spectrometry. Far-UV (200–260 nm) circular
dichroism (CD) spectra were obtained at 30 ꢀC on a
J-720W spectropolarimeter (Jasco, Tokyo). The sample
contained proteins at 0.52 mg/ml (0.33 mM) in 10 mM
MOPS buffer (pH 7.2). The optical path of the quartz
cell was 2 mm. The mean molar ellipticity, ꢃ [deg
cm2 dmolꢁ1], was calculated using an average molecular
weight of 110 for one amino acid residue.15)
When GDOxp was overproduced in E. coli BL21
(DE3) cells harboring pET25b-GDOxp, it formed most-
ly an insoluble protein (Fig. 2A). We expected that a
molecular chaperon would help GDOxp to fold properly
to take soluble form. In fact, the co-expression systems
of pET25b-GDOxp and pSTV28-GroES/EL in E. coli
were successful in recovering, about half of the GDOxp
in the soluble fraction. Soluble GDOxp was purified

Gentisate 1,2-Dioxygenase from Xanthobacter polyaromaticivorans 127W
195
Fig. 1. Amino Acid Sequence Alignment of Selected GDO and HNDO.
GenBank accession numbers are as follows: DbdB (GDOxp), gentisate 1,2-dioxygenase from X. polyaromaticivorans 127W (AB121977);
DtdA-PAO, gentisate 1,2-dioxygenase from P. aeruginosa (AE004674); XlnE, gentisate 1,2-dioxygenase from P. alcaligenes (AF173167);
PhdI, 1-hydroxy 2-naphthoic acid dioxygenase from Nocaridoides sp. KP7 (D89987). Consensus amino acid residues among cupin family
proteins are shown under the alignment. Cupin motifs are indicated by thick boxes in the N-terminal domain and by broken boxes in the putative
C-terminal domain. Four histidine residues essential for enzyme activity7) are shaded in gray. The secondary structure of GDOxp was predicted
by the Jpred program,9) and the results are shown by boxes (alpha-helix) and arrows (beta-sheet) under the sequence alignment. The external
alpha-helix region and the N-terminal and C-terminal cupin domains are discriminated by dotted, open, and shaded symbols respectively.
A
B
kDa
1
2
3
4
kDa
3
kDa
1
2
4
97
66
97
66
97
66
45
45
45
30
30
30
20.1
20.1
20.1
14.4
14.4
14.4
Fig. 2. SDS–PAGE Analyses of GDOxp.
Electrophoresis was performed with 15% polyacrylamide gel in the presence of SDS, and the gel was stained with Coomassie brilliant blue. A,
Lanes 1 and 2 show soluble and insoluble fractions obtained from E. coli BL21 (DE3) cells transformed with pET25b-GDOxp, respectively.
Lanes 3 and 4 are soluble and insoluble fractions from E. coli BL21 (DE3) cells harboring both pET25b-GDOxp and pSTV28-GroES/EL,
respectively. Phosphorylase b (97,000), albmin (66,000), ovalbumin (45,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and alpha-
lactalbumin (14,400) were used as molecular standard markers. GroEL and GDOxp protein bands are indicated by arrows. B, Comparison of the
purity of GDOxp by SDS–PAGE. Lane 1, total soluble proteins prepared from E. coli BL21 (DE3) cells transformed by pET25b-GDOxp/
pSTV28-GroES/EL. Lane 2, after ammonium fractionation. Lane 3, after purification by anion exchange column (HitrapQ) chromatography.
Lane 4, after gel filtration column (Superdex200pg) chromatography.

196
S. HIRANO et al.
Table 1. Relative Activity of GDOxp against Several Substrates
through three purification steps to apparent homogeneity
by SDS–PAGE analysis (Fig. 2B). Finally, 5.6 mg of
GDOxp was purified from 1 liter of culture. The specific
activity of the purified enzyme was 102 units/mg (pH
7.5). The molecular mass of GDOxp was estimated to
be 40 kDa on SDS–PAGE, comparable to the value cal-
culated from its deduced amino acid sequence, 39.3 kDa.
When GDOxp was analyzed by gel filtration chroma-
tography, it eluted at a position corresponding to a
molecular weight of 146 kDa, suggesting that GDOxp
formed a homotetramer. It has been reported that GDOs
require ferrous ion for their catalytic activity.6) The
ferrous ion content was measured and the amount of
ferrous ion bound to 1 mol subunit of GDOxp was
determined to be 0.98 mol. This result suggests that only
one of the cupin domains in GDOxp, probably the first
one, was functional as an active ligand for ferrous ion.
Substrate
relative activity (%)
Gentisate
Catechol
Benzoate
100a
0
0
0
5-Methylsalicylate
5-Chlorosalicylate
5-Aminosalicylate
0
5
Homogentisate
1-Hydroxy 2-naphthoate
1,4-Dihydroxy2-naphthoate
0
2.9
102.8
aThe activity against gentisate was considered to be 100%.
against 1,4-dihydroxy-2-naphthoate, but only in part
(0.3–11% active).1) In contrast to these GDOs, GDOxp
was fully active against 1,4-dihydroxy-2-naphthoate.
GDO from P. alcaligenes NCIB9867, the closest rela-
tive of GDOxp, showed even higher activity (127%)
against 3-methylgentisate than gentisate, but the avail-
ability of 1,4-dihydroxy-2-naphthoate to the enzyme is
unknown.18) It should also be noted that HNDO has been
reported to be inactive against gentisate.11) The Km and
kcat values of GDOxp for 1-hydroxy-2-naphthoate were
determined to be 20:9 ꢂ 3:6 mM and 33:9 ꢂ 2:6 minꢁ1
siteꢁ1 respectively. These values suggest that the low
activity of GDOxp for 1-hydroxy-2-naphthoate was due
to a reduction in the turnover rate rather than to poor
affinity of the enzyme for the bulky hydroxyl naph-
thoate. The activity of GDOxp against gentisate was
reduced to 18.5% in the presence of equimolar (0.33
mM) of 1-hydroxy 2-naphthoic acid, and 38% by sal-
icylate, respectively. Comparable Km values for GDOxp
against 1-hydroxy-2-naphthoate and gentisate indicate
that the structure of the substrate binding site of GDOxp
is more flexible than other GDOs, flexible enough to
accept both mono- and polycyclic aromatic hydrocar-
bons as substrates. A unique salicylate dioxygenase
(AAQ91293) has been reported from Ps. salicylatox-
idans that is active against salicylate variants. It showed
highest activity against 1-hydroxy-2-naphthoate.19) The
enzyme showed 19% and 18% activity against salicylate
and gentisate respectively as compared with its activity
against 1-hydroxy-2-naphthoate. Salicylate dioxygenase
from Ps. salicylatoxidans and GDOxp shared 33%
amino acid sequence identity, suggesting evolutional
distance.
Kinetic properties
Initial reaction rates were measured at different
concentrations of gentisate (3.3 mM–0.5 mM). Under
these conditions, the enzyme followed Michaelis–
Menten kinetics. Km and kcat values were determined
to be 18:6 ꢂ 1:6 mM and 5;430 ꢂ 330 minꢁ1ꢃsiteꢁ1 re-
spectively. The Km and kcat values of GDO from
P. acidovorans and Comamonas testosteroni for genti-
sate have been reported to be 74 mM and 19,300
minꢁ1 siteꢁ1, and 85 mM and 22,000 minꢁ1 siteꢁ1, re-
spectively.12) X. polyaromaticivorans 127W is capable
of degrading PAHs and HACs under ELO conditions.
The cells maintained 23.5% of dibenzothiophene de-
grading activity even under an oxygen condition of
6.7 mM (0.2 ppm) as compared to the aerobic condition,
218.8 mM (6.5 ppm).7) The Km and Kcat values of GDOxp
for oxygen were determined to be 19:2 ꢂ 2:4 mM and
4;420 ꢂ 230 minꢁ1 siteꢁ1 respectively. The values for
GDO from P. acidovorans and C. testosteroni for oxy-
gen are 55 mM and 19,700 minꢁ1 siteꢁ1 and 96 mM and
38,500 minꢁ1 siteꢁ1 respectively.12) We do not know
whether GDOxp is a rate-limiting enzyme in the
degradation pathway, but lower Km values of GDOxp
against oxygen than other GDOs would help the cells to
metabolize PAHs and HACs effectively under ELO
conditions.
Substrate specificity
The oxygenation activity of GDOxp against gentisate
and salicylate substitutes was examined spectrophoto-
metrically. Substrates of 1-hydroxy-2-naphthoate dioxy-
genase (HNDO), 1-hydroxy-2-naphthoate, and 1,4-dihy-
droxy-2-naphthoate were also tested for the enzyme
assay because GDOxp shared significant amino acid
sequence similarity (33% identical) with HNDO.
Among the compounds tested, gentisate and 1,4-dihy-
droxy-2-naphthoate were the most preferred, and 5-
aminosalicylate was a partly preferred substrate for
GDOxp (Table 1). GDOs from P. testosteroni and
P. acidovorans also have been found to be active
Stability of GDOxp
GDOxp lost its activity after being kept in buffers
below pH 6.5 or above 8.0 for 12 h in the absence of
ferrous ion and cysteine. In order to shed light on this
unusual instability of GDOxp against pH, GDOxp,
which had been dialyzed against 10 mM phosphate
buffer (pH 9.0) for 12 h, was analyzed as to its structure
by gel filtration chromatography. It was found that most
of the protein existed as inactive monomers (39 kDa),
and that these monomer proteins did not contain ferrous

Gentisate 1,2-Dioxygenase from Xanthobacter polyaromaticivorans 127W
197
A
B
140
120
100
80
4,000
0
200
220
240
260
-4,000
60
40
-8,000
20
0
-12,000
0
20
40
60
Wave length (nm)
Incubation time (min)
Fig. 3. Heat Treatment of GDOxp.
A, Reduction in the activity after heat treatment is shown. GDOxp (4.3 mM) in 10 mM MOPS buffer (pH 7.2) containing 10% glycerol was
incubated at 46 ꢀC (closed circle), 50 ꢀC (closed square), or 55 ꢀC (closed triangle) for appropriate times. A portion of the incubation sample was
taken at the indicated times and the activity was assayed at 30 ꢀC. GDOxp incubated at 50 ꢀC in the presence of 20 mM ferrous ion is also shown
(open square). B, CD spectra of GDOxp with no incubation (thick line) and after incubation for 1 h at 50 ꢀC (thin line) were measured using
J-720W.
ion (0.06 mol/mol subunit). GDOxp was also heat-
unstable without ferrous ion (Fig. 3A). It lost almost all
0.30
its activity after incubation at 50 ꢀC for 15 min. How-
ever, far-UV CD spectra suggested that there was no
significant change in the secondary structure of GDOxp
0.25
0.20
even after incubation at 50 ꢀC over 1 h (Fig. 3B). When
the ꢃ value at 218 nm was monitored while increasing
0.15
the temperature at a rate of 1 ꢀC/min, a dramatic ir-
reversible change was observed between 70 and 80 ꢀC.
These results suggest that the backbone structure of
0.10
0.05
0.00
GDOxp, including the cupin domains, is rather heat-
stable.
-0.05
0
2
4
6
8
10
Role of ferrous ion in GDO activity and structure
formation
Concentration of Fe 2+ (µM)
apo-GDOxp was prepared by a treatment of purified
GDOxp (holo-GDOxp) with 10 mM EDTA and dialysis
against buffer A to remove ferrous ion and EDTA. When
apo-GDOxp was analyzed by gel filtration chromatog-
raphy, most of the protein eluted at a position corre-
sponding to inactive monomer protein without binding
to ferrous ion (0.05 mol/mol subunit).
Fig. 4. Effect of Ferrous Ion on the Reactivation of Monomer Apo-
GDOxp.
The activity of each 30.4 nM monomer apo-GDOxp was measured
at different ferrous ion concentrations. The curve was fitted to the
Hill equation.13)
It was also found that inactive apo-GDOxp could be
reactivated in the buffer containing ferrous ion. The
initial reaction rates of apo-GDOxp under different
ferrous ion concentrations exhibited sigmoidal curves
(Fig. 4, Hill coefficient = 5.0). These results suggest
that ferrous ion acted as a cooperative effector to form
an active tetramer holo-enzyme. In order to test this
possibility, the monomer apo-GDOxp was purified by
gel filtration chromatography, concentrated to 0.65 mg
in 2 ml buffer, and applied to the gel filtration column
again with and without the addition of 80 mM ferrous
ion. When ferrous ion was added to the fraction, about
90% of GDOxp was eluted as higher molecular weight
species corresponding to a tetramer. Moreover, 68% of
the full activity was restored in this fraction, and the
tetramer GDOxp contained 0.95 mole ferrous ion/mole
subunit. The sample with no addition of ferrous ion was
eluted again at the position of the monomer species. No
reconstitution was observed with the addition of 2 mM
cysteine or other divalent cations such as Zn2þ, Co2þ
,
Ni2þ, Mn2þ, and Ca2þ. GDOxp was completely inacti-
vated by 2 mM of ferric ion, and the addition of 1 mM
copper ions formed a visible precipitate. These results
indicate that ferrous ion specifically induces tetrameri-
zation and activation of monomer apo-GDOxp.
Limited chymotryptic digestion of GDOxp
In order to analyze the domain structure of GDOxp, it

198
S. HIRANO et al.
sequence of 37.6 kDa protein was determined to be
holo-GDOxp
10
apo-GDOxp
10
A
T30VLNNI, while that of 20.5 kDa protein was not
determined. Because apo-GDOxp consists mainly of
monomer protein, a hypothesis is that the flanking
region of this sequence is one of the interfaces of the
tetramer structure of GDOxp. We constructed another
expression plasmid, pET25b-dGDOxp, for the produc-
tion of truncated GDOxp in the N-terminal peptide
(from S2 to W29) region. Heterologously produced
dGDOxp formed monomers, although it contained
1.2 mol ferrous ion/mol protein. Monomeric dGDOxp
showed no activity against gentisate. These results
suggest that the N-terminal region is important for
tetramerization, and also that the tetramerization of
GDOxp is essential to form an active structure.
(min)
1
5
0
1
5
GDOxp
37.6 kDa
20.5 kDa
23.2 kDa
16.1 kDa
B
In this study, we found that GDOxp was fully
functional against 1,4-dihydroxy-2-naphthoate as well
as gentisate. Further experiments are necessary to
demonstrate that GDOxp is a bi-functional enzyme in
the PAHs and HACs degradation pathways of strain
127W. Monomer and tetramer structures were reversi-
ble. GDOxp formed an active tetramer structure by
binding with ferrous ion in a cooperative manner.
Fig. 5. Analysis of Domain Structure of GDOxp.
A, SDS–PAGE analysis of chymotryptic digestion at different
treament times. The reaction was performed at 37 ꢀC in 10 mM
MOPS (pH 7.2) containing 2.5 mM DTT for 0 to 10 min at a ratio of
chymotrypsin/GDOxp = 1:100 (w/w). Samples were boiled for
1 min to stop the reaction at the indicated times, and analyzed on
15% SDS–polyacrylamide gel. Arrows indicate 23.2 kDa and 16.1
kDa products corresponding to N-terminal and C-terminal cupin
domains respectively. B, The position of cleavage by chymotrypsin
is shown schematically. The 23.2 kDa and 16.1 kDa proteins were
produced by cleavage between Y199 and G200. The 36.7 kDa
protein was truncated at N-terminal 30 amino acids.
Acknowlegment
This work was supported in part by the Institute for
Fermentation, Osaka, Japan.
References
was partially digested with chymotrypsin. Peptide bonds
between domains are generally more susceptible to
proteolytic digestion than those within a domain, and
limited proteolysis is a useful method to disconnect
domains from one another. The time course of chy-
motrypsin digestion of GDOxp is shown in Fig. 5.
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structural domains in GDOxp. The N-terminal sequence
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structure from 179–229 (Fig. 1). The C-terminal domain
containing putative motif 3 and motif 4 should form a
rigid structure like the N-terminal domain containing
motif 1 and motif 2.
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