Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans Strain CA10

Article abstract of DOI:10.1271/bbb.67.36

2-Hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-dienoic acid [6-(2′-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6- oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a K_m of 2.51 μM and k_cat of 2.14 (s^-1) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25°C). The effect of the presence of an amino group or hydroxyl group at the 2′-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2′-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2′- hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2′-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.

Full text of DOI:10.1271/bbb.67.36

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Purification and Characterization of meta-Cleavage
Compound Hydrolase from a Carbazole Degrader
Pseudomonas resinovorans Strain CA10
Hideaki NOJIRIa, Hiroko TAIRAa, Kenichi IWATAa, Kenichi MORIIa, Jeong-Won NAMa, Takako
YOSHIDAa, Hiroshi HABEa, Shugo NAKAMURAb, Kentaro SHIMIZUb, Hisakazu YAMANEa &
Toshio OMORIa
a Biotechnology Research Center, The University of Tokyo 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan
b Department of Biotechnology, The University of Tokyo 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan
Published online: 22 May 2014.
To cite this article: Hideaki NOJIRI, Hiroko TAIRA, Kenichi IWATA, Kenichi MORII, Jeong-Won NAM, Takako YOSHIDA, Hiroshi
HABE, Shugo NAKAMURA, Kentaro SHIMIZU, Hisakazu YAMANE & Toshio OMORI (2003) Purification and Characterization
of meta-Cleavage Compound Hydrolase from a Carbazole Degrader Pseudomonas resinovorans Strain CA10, Bioscience,
Biotechnology, and Biochemistry, 67:1, 36-45, DOI: 10.1271/bbb.67.36
To link to this article: http://dx.doi.org/10.1271/bbb.67.36
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Biosci. Biotechnol. Biochem., 67 (1), 36–45, 2003
Puriˆcation and Characterization of meta-Cleavage Compound Hydrolase
from a Carbazole Degrader Pseudomonas resinovorans Strain CA10
1
Hideaki NOJIRI,1 Hiroko TAIRA,1 Kenichi IWATA,1 Kenichi MORII
,
Jeong-Won NAM,1 Takako YOSHIDA,1 Hiroshi HABE,1 Shugo NAKAMURA
,
2
1,
†
Kentaro SHIMIZU,2 Hisakazu YAMANE,1 and Toshio OMORI
2
1Biotechnology Research Center and Department of Biotechnology, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received May 14, 2002; Accepted September 24, 2002
2-Hydroxy-6-oxo-6-(2
?-aminophenyl)-hexa-2,4-
resinovorans strain CA10 was isolated from a sample
of activated sludge as a bacterium that can use carba-
zole as the sole source of carbon, nitrogen, and
energy.6) The initial step of carbazole degradation by
strain CA10 is dioxygenation at the angular position
adjacent to the nitrogen atom to give the dihydrox-
ylated intermediate, which is spontaneously convert-
dienoic acid [6-(2 -aminophenyl)-HODA] hydrolase,
?
involved in carbazole degradation by Pseudomonas
resinovorans strain CA10, was puriˆed to near
homogeneity from an overexpressing Escherichia coli
strain. The enzyme was dimeric, and its optimum pH
was 7.0–7.5. Phylogenetic analysis showed the close
relationship of this enzyme to other hydrolases involved
in the degradation of monocyclic aromatic compounds,
and this enzyme was speciˆc for 2-hydroxy-6-oxo-6-
phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having
little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic
acid and 2-hydroxymuconic semialdehyde. The enzyme
ed to 2?-aminobiphenyl-2,3-diol. This process is
called angular dioxygenation. Then,
2?-amino-
biphenyl-2,3-diol is converted to anthranilate and 2-
hydroxypenta-2,4-dienoate via meta-cleavage and
subsequent hydrolysis. 2-Hydroxypenta-2,4-dienoate
is metabolized to TCA cycle intermediates through
the meta-cleavage pathway.
had a Km of 2.51
HODA (50 m sodium phosphate, pH 7.5, 25
eŠect of the presence of an amino group or hydroxyl
group at the 2 -position of phenyl moiety of 6-phenyl-
HODA on the enzyme activity was found to be small;
the activity decreased only in the order of 6-(2
aminophenyl)-HODA (2.44 U mg) 6-phenyl-HODA
-hydroxyphenyl)-
hexa-2,4-dienoic acid (1.05 U mg). The eŠects of 2
m
M
and kcat of 2.14 (s－1) for 6-phenyl-
C). The
M
9
By shotgun cloning and gene walking, we earlier
isolated the carAaAaBaBbCAcAdDFE gene cluster
involved in the conversion of carbazole to anthrani-
late and 2-hydroxypenta-2,4-dienoate, and of 2-
hydroxypenta-2,4-dienoate to TCA cycle intermedi-
ates.7–9) The terminal oxygenase component, CarAa,
?
?
-
À
W
(1.99 U mg)
À2-hydroxy-6-oxo-6-(2?
of the angular dioxygenase system has less than 10
z
W
?-
identity to the catalytic subunits of oxygenase com-
ponents of known dioxygenase systems, although
W
substitution on the activity were in accordance with the
predicted reactivity based on the calculated lowest unoc-
cupied molecular orbital energy for these substrates.
identity was 39 with OxoO protein in the 2-oxo-
z
1,2-dihydroquinoline 8-monooxygenase system,10) as
reported by Nam et al.
11) The meta-cleavage pathway
Key words: meta-cleavage compound hydrolase; car-
bazole; dibenzofuran; Pseudomonas
resinovorans strain CA10
enzymes, CarD, CarE, and CarF, had about 70–80
z
identity with reported isofunctional enzymes.9) Car-
BaBb (the meta-cleavage enzyme) and CarC [the
meta-cleavage compound hydrolase; 2-hydroxy-6-
Carbazole is a toxic
N
-heterocyclic aromatic com-
oxo-6-(2?-aminophenyl)-hexa-2,4-dienoic acid (2?-
pound containing dibenzopyrrole system, and is der-
ived from coal tar and shale oil.1,2) EŠorts have been
made to isolate the carbazole-degrading bacteria and
to analyze the microbial metabolism of this com-
pound as reviewed previously.3–5) Pseudomonas
aminophenyl-HODA) hydrolase] had about 30
z
identities to known isofunctional enzymes.8) These
results suggest that the meta-cleavage enzyme and
subsequent metabolic enzymes, CarBaBbCDEF, may
be more closely related to the corresponding enzymes
†
To whom correspondence should be addressed. Fax: +81-3-5841-8030; E-mail: aseigyo
6-(2 -aminophenyl)-HODA, 2-hydroxy-6-oxo-6-(2 -aminophenyl)-hexa-2,4-dienoic acid; HODA, 2-hydroxymuconic
-hydroxyphenyl)-HODA, 2-hydroxy-6-oxo-6-(2 -hydroxyphenyl)-hexa-2,4-dienoic acid; IPTG, isopropyl- -thiogalac-
＠
mail.ecc.u-tokyo.ac.jp
Abbreviations
semialdehyde; 6-(2
:
?
?
?
?
b-D
topyranoside; LUMO, lowest unoccupied molecular orbital; 6-methyl-HODA, 2-hydroxy-6-oxohepta-2,4-dienoic acid; 6-phenyl-HODA, 2-
hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid

C-C Bond Hydrolase from a Carbazole Degrader
37
Fig. 1. Phylogenetic Tree of the meta-Cleavage Compound Hydrolases.
The tree was constructed by the neighbor-joining method with the PHYLIP package (version 3.573; obtained from J. Felsenstein,
University of Washington). The bootstrap values of 80 or more from 100 resamplings are shown for each node. The scale bar denotes
0.5 substitution per site. The name of bacterial strain or plasmid and the DDBJ EMBL GenBank accession number are shown in paren-
W
W
theses after the enzyme name. Grouping reported by Herna
á
ez et al
.
22) are shown by the term, I, II, III, and IV. CarC from P. resinovo-
rans strain CA10 is shaded.
in degradation pathways for other aromatic com-
pounds than CarAa. Dibenzofuran and dibenzo-
tion of monocyclic and bicyclic aromatic compounds
are in groups III and I, respectively,22) although there
are several hydrolases with intermediate features.
The substrate speciˆcity of CarC was similar to that
of hydrolases classiˆed into group I, but a closer
phylogenetic relation was observed with those in
group III. Although classiˆed into group III, CarC
of strain CA10 and related proteins (CarC of Sphin-
gomonas sp. strain GTIN1123) and DxnB of S.
wittichii strain RW124,25)) have intermediate features
and comprise a highly divergent branch. With their
diŠerent substrate speciˆcities, these enzymes might
be classiˆed into a new group apart from group III.
These facts suggest that CarC of strain CA10 is a
novel hydrolase. Thus, it is interesting to discover the
structure and function of CarC, and to compare
them with those of other meta-cleavage compound
hydrolases involved in the degradation pathways of
monocyclic, bicyclic, and heterocyclic aromatic com-
pounds. With these considerations, we puriˆed and
characterized CarC. On the basis of enzymatic fea-
tures, we discuss the relationship between CarC and
other hydrolases.
p
-
dioxin, the parental compounds of dioxin, which
causes environmental problems, were degraded
through a pathway similar to that of carbazole by
strain CA10.3,5,12) This similarity may re‰ect the
relatedness of these two degradation pathways. In
fact, carbazole-degrading enzymes degrade diben-
zofuran, dibenzo-
p-dioxin, and their chlorinated
derivatives.7,13,14)
The substrate speciˆcity of meta-cleavage com-
pound hydrolase helps to govern the overall selectivi-
ty of the pathway with respect to the degradation of
aromatic compounds.15–20) It has been proposed that
CarC is one of the key determinants of substrate
speciˆcity of the carbazole degradation system of
strain CA10 for chlorinated dibenzofuran and chlori-
nated dibenzo-p
-dioxin.14) Sequence comparisons of
such hydrolases indicate that they belong to the fami-
ly of proteins that have the a b hydrolase fold,21) and
W
that the hydrolases can be divided into several groups
22)

as indicated by Hernaez et al
.
(Fig. 1). Generally
this phylogeny-based grouping well corresponds to
the grouping based on the preferred substrate, espe-
cially for the size of the functional group at C6 of the
substrate. Most hydrolases involved in the degrada-

38
H. NOJIRI et al.
purity of CarC was checked by SDS-PAGE30) with a
13 polyacrylamide gel. The gels were stained for
proteins with Coomassie Brilliant Blue R-250. The
protein concentration was measured by the method
of Bradford31) with bovine serum albumin as the stan-
dard and with a Protein Assay Kit II (Bio-Rad
Laboratories, Richmond, CA).
Materials and Methods
z
Bacterial strains, plasmids, media, and culture
conditions. Esherichia coli strain JM10926) was used
as the host of the plasmids pUC118,26) pUC119,26)
pSTV28 (Takara Shuzo Co., Ltd., Kyoto, Japan), or
their derivatives. The pSTV28-based plasmid pIP301
containing a 2.4-kb SalI-EcoRI fragment encoding
CumC (meta-cleavage enzyme involved in isopropyl-
benzene degradation in Pseudomonas ‰uorescens
strain IP01) was prepared from pIP103.27) E. coli
strains were grown on 2xYT medium28) with ampicil-
Preparation of substrates for assays of activity of
CarC.
2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid (6-phenyl-HODA; a meta-cleavage compound in
the biphenyl degradation pathway) was prepared
from 2,3-dihydroxybiphenyl with E. coli cells
harboring pUCA122 as described previously.8) 2-
Hydroxy-6-oxohepta-2,4-dienoic acid (6-methyl-
HODA; a meta-cleavage compound in the toluene
degradation pathway) and 2-hydroxymuconic semial-
dehyde (HODA; a meta-cleavage compound in the
catechol degradation pathway) were similarly pre-
pared from 3-methylcatechol and catechol, respec-
tively, with E. coli cells harboring pIP301.
lin (50
37 C. For plate cultures, media solidiˆed with 1.6
agar (wt vol) were used. An appropriate amount of
mg ml) or chloramphenicol (50 mg ml) at
W
W
9
z
W
isopropyl-
b- -thiogalactopyranoside (IPTG) was
D
added to the medium to induce the expression of the
enzymes as described below.
DNA manipulation. Plasmid DNA was prepared
from the E. coli host strain by alkaline lysis.29) Res-
triction endonuclease and a DNA Ligation Kit ver-
sion 2 (Takara Shuzo) were used according to the
manufacturer's instructions. DNA fragments were
Assay of activity of puriˆed CarC. The hydrolase
activity of the puriˆed CarC was assayed by monitor-
fractionated by electrophoresis with 0.9
z
Agarose S
ing of changes in A434, A388, and A375 with the
(Nippon Gene Co., Ltd., Tokyo, Japan) in TAE
buŠer,28) and were extracted from the gel with Con-
cert Rapid Gel Extraction System (Invitrogen Corp.,
Carlsbad, CA) by the manufacturer's instructions.
Other DNA manipulations were done by standard
methods.28)
substrates, 6-phenyl-HODA, 6-methyl-HODA, and
HODA, respectively, with a Beckman DU-7400 spec-
trophotometer equipped with
a thermojacketed
cuvette holder and a circulating water bath. The
molar extinction coe‹cients of 6-phenyl-HODA, 6-
methyl-HODA, and HODA under the assay condi-
tions used (including a pH of 7.5) were taken to be
34)
Expression and puriˆcation of CarC. E. coli strain
JM109 harboring pUCA1918) containing a 1.3-kb
HindIII-SphI fragment encoding CarC was grown on
100 ml of 2xYT medium containing ampicillin and
13,200,32) 32,000,33) and 36,000
M
„1 cm„1
,
respec-
tively. The standard assay was done in a total volume
of 1 ml of 50 m sodium phosphate buŠer (pH 7.5)
at 25 C. To estimate the relative activity of CarC
M
9
IPTG (ˆnal concentration, 0.5 m
incubation at 37 C, the cells were harvested, washed
twice with 50 m sodium phosphate buŠer (pH 7.5;
M
). After 18 h of
toward 6-phenyl-HODA, 6-methyl-HODA, and
HODA, a substrate was added to the ˆnal concentra-
9
M
tion of 40
of m, 6-phenyl-HODA was added to the ˆnal con-
centration of 1 to 80 . The reaction was started by
the addition of 10 g of puriˆed enzyme if not men-
tioned otherwise. One unit of activity was deˆned as
the amount of the enzyme that degraded 1 mol of
substrate per minute at 25 C. Relative activities are
expressed as percentages of that measured with 6-
phenyl-HODA as the substrate, taken to be 100
m
M
to the reaction mixture. For calculation
buŠer A), and resuspended in 30 ml of the same
buŠer. The cell suspensions were sonicated and cen-
K
m
M
×
trifuged at 21,600
pernatants were used as crude cell extracts. Puriˆca-
tion was done at 4 C throughout. The crude cell
extract was brought to 40 saturation with solid
g
at 4
9
C for 60 min, and the su-
m
9
m
z
9
ammonium sulfate. The mixture was stirred for
30 min, and the precipitate was discarded. The
resultant supernatant was dialyzed against buŠer A.
From the solution after the dialysis, CarC was sepa-
rated on a column of DEAE-Sephadex A-50 (Amer-
sham Pharmacia Biotech UK Ltd., Little Chalfont,
Buckinghamshire) with a linear gradient of 0.0 to
z.
To discover the eŠects of pH on the enzyme activi-
ty, we assayed the hydrolase activity toward 6-
phenyl-HODA in the following buŠers at 50 m : so-
M
dium citrate buŠer (pH 5.0–6.0), sodium-potassium
phosphate buŠer (pH 5.0–7.0), sodium phosphate
buŠer (pH 6.0–8.0), and Tris-HCl buŠer (pH
7.5–9.0). As molar extinction coe‹cients at various
0.5
of Sephacryl S-200 (Amersham Pharmacia Biotech
UK) equilibrated with buŠer A containing 0.1
M
NaCl. CarC was further puriˆed on a column
M
pHs, the values reported by Hatta et al.
35) were used.
NaCl. The relative molecular weight of the native
protein was estimated by calibrated gel ˆltration
chromatography on a Sephacryl S-200 column. The
To discover the eŠects of temperature, after CarC
was incubated at various temperatures for 1 to
10 min, the remaining hydrolase activity for 6-

C-C Bond Hydrolase from a Carbazole Degrader
39
phenyl-HODA was assayed at 25
above.
9
C as described
used as a substrate. To 1 ml of 50 m
buŠer (pH 7.5), the 10 to 20
solution of 2
2,3-dihydroxybiphenyl was added to the ˆnal concen-
tration of 100 . Two micrograms of the puriˆed
M
Tris-HCl
m
l of an ethanol stock
-substituted-2,3-dihydroxybiphenyl or
?
Preparation of 2
?
-substituted 2,3-dihydroxybi-
phenyl. -Aminobiphenyl-2,3-diol and 2,2
2
?
?,3-tri-
m
M
hydroxybiphenyl were prepared from carbazole and
dibenzofuran, respectively, by a resting cell reaction
with E. coli cells harboring pUCARA expressing
angular dioxygenase (CarAaAcAd) as described
previously.7) After the conversion of the substrate to
CarB was added to this solution, and the mixture was
stirred for 30 s, resulting in the complete conversion
of the starting compounds to the corresponding 6-
(2?-substituted phenyl)-HODA. Then, 40 mg of CarC
was added to the reaction mixture, and changes in
speciˆc absorption (see Results section) were moni-
tored. The molar extinction coe‹cients found in this
study were used. The measurements were done at
2?-substituted-2,3-dihydroxybiphenyl, the reaction
mixtures were extracted with an equal volume of
ethyl acetate. The ethyl acetate layers were dried over
Na2SO4 and then evaporated to dryness at room tem-
perature under the reduced pressure. The residues
were dissolved in a small amount of ethyl acetate and
put on a silica gel column for chromatography as
25
unit of activity was deˆned as the amount of the en-
zyme that degraded 1 mol of substrate per minute at
25 C.
To analyze the enzymatic and nonenzymatic con-
9C, with the equipment mentioned above. One
m
9
described previously.13) The puriˆed
2
?
-amino-
biphenyl-2,3-diol and 2,2
?
,3-trihydroxybiphenyl
versions of HODA-related compounds, we extracted
reaction mixtures containing CarB with or without
CarC with equal volumes of ethyl acetate under acid-
ic conditions (pH 2–3). The organic phase was dried
over Na2SO4, and then evaporated to dryness under
reduced pressure. The resultant extract was analyzed
were dissolved in a small amount of ethanol. The
concentrations of these compounds in the resultant
solution were measured by monitoring of the oxygen
consumption during complete meta-cleavage with an
excess of the puriˆed CarB36) with a Clark-type
oxygen electrode (Iijima Electronics Co., Aichi,
Japan) as described previously.8)
by GC-MS after the trimethylsilylation with
methyl- -trimethylsilyltri‰uoroacetamide (70 C for
20 min) as described previously.13)
N-
N
9
Measurment of molar extinction coe‹cient of
meta-cleavage compounds from 2
?
-substituted-2,3-
Tris-HCl
buŠer (pH 7.5), an excess of puriˆed CarB and a
known amount of 2 -aminobiphenyl-2,3-diol or
2,2 ,3-trihydroxybiphenyl were added, resulting in
the formation of 2-hydroxy-6-oxo-6-(2 -amino-
phenyl)-hexa-2,4-dienoic acid [6-(2 -aminophenyl)-
HODA; a meta-cleavage compound in the carbazole
degradation pathway] or 2-hydroxy-6-oxo-6-(2
hydroxyphenyl)-hexa-2,4-dienoic acid [6-(2 -hydro-
Theoretical calculations. Theoretical calculations
were done by the semiempirical quantum-mechanical
AM1 method37) in the MOPAC 2000 program
(Fujitsu Ltd., Tokyo, Japan). In the description of
the solvent, we used the COSMO method.38) All
structures were optimized with the eigenvector by the
method of Baker.39)
dihydroxybiphenyl. To 1 ml of 50 m
M
?
?
?
?
?-
Results
?
xyphenyl)-HODA; a meta-cleavage compound in the
dibenzofuran degradation pathway]. After complete
meta-cleavage, the absorbance between 190 to
800 nm of the reaction mixture was monitored at
Expression and puriˆcation of CarC
CarC was su‹ciently expressed for puriˆcation
and enzymatic characterization from E. coli cells
harboring the pUC119-based plasmid pUCA1918)
(Fig. 2). IPTG was added to the ˆnal concentration
25
9
C as described above.
The stability of 6-phenyl-HODA and 6-(2
?
-substit-
of 0.5 m at the same time as the seeding of an over-
M
uted phenyl)-HODAs was measured by spectro-
night ˆrst culture, because no diŠerence was detected
when IPTG was added when the cell growth reached
an OD550 of 1.0. Eventually, 34.6 mg of CarC was
puriˆed from a 200-ml culture of these E. coli cells
(Table 1). CarC was apparently puriˆed to near
homogeneity, as indicated by SDS-PAGE (Fig. 2),
and its relative molecular mass was estimated to be
31,000 Da, close to the value deduced from the
amino acid sequence (31,330 Da). The relative
molecular weight of the native protein was estimated
by gel ˆltration chromatography, to be 70,000.
photometric monitoring of changes in the absor-
bance of a solution of these compounds (50 m
M
Tris-
HCl buŠer, pH 7.5, 25 C) at the wavelength of
9
maximum absorbance. Half-lives were calculated by
ˆtting of the data to ˆrst-order decay.
Assay of hydrolase activity for 6-(2
phenyl)-HODAs. The hydrolase activity of CarC for
6-(2 -substituted phenyl)-HODAs was measured by
monitoring of the conversion of 2 -substituted-2,3-
dihydroxybiphenyl to 2 -substituted benzoic acid and
?-substituted
?
?
?
2-hydroxypenta-2,4-dienoate with puriˆed CarB and
CarC. For comparison, 2,3-dihydroxybiphenyl was

40
H. NOJIRI et al.
Table 1. Puriˆcation of CarC Protein from 200-ml Culture of E. coli Harboring pUCA191
M
Enzymatic activity for 6-phenyl-HODA was monitored throughout puriˆcation. Hydrolase activity was assayed at 259C in 50 m sodium
phosphate buŠer (pH 7.5). One unit of enzyme activity is the activity that converts 1
m
mol of 6-phenyl-HODA.
Total protein
(mg)
Total activity
(U)
Speciˆc activity
Puriˆcation
(fold)
Puriˆcation step
Crude extract
Supernatant after precipitation
DEAE-Sephadex A-50
Sephacryl S-200
(U mg)
W
136
233
202
175
133
1.71
2.76
3.52
3.84
1.00
1.61
2.06
2.25
73.3
49.8
34.6
aminophenyl)-HODA and 6-(2?-hydroxyphenyl)-
HODA had wavelengths of maximum absorbance at
403 and 438 nm, respectively. The extinction
coe‹cients at the appropriate wavelength for freshly
prepared 6-(2
hydroxyphenyl)-HODA were 9,140 and 22,100
cm„1, respectively. Half-lives of 6-(2
-aminophenyl)-
HODA and 6-(2 -hydroxyphenyl)-HODA under the
?-aminophenyl)-HODA and 6-(2?-
„1
M
?
?
conditions we used were 6.1 and 7.3 min, respec-
tively; that for 6-phenyl-HODA is 58 h.19)
EŠects of substitution at 2
moiety on hydrolase activity of CarC
Because of the too-ready degradation of 6-(2
stituted phenyl)-HODAs, we measured the hydrolase
activity of CarC for these compounds by a combina-
?-position of phenyl
?-sub-
Fig. 2. SDS-PAGE of Samples from the Subsequent Puriˆcation
Steps.
Lane 1, molecular mass standards; 2, crude cell extract; 3, su-
tion assay in which the substrates, 6-(2
?-substituted
pernatant after precipitation with 40
z (wt vol) (NH4)2SO4; 4,
W
phenyl)-HODAs, were produced by the meta
-
preparation after DEAE-Sephadex A-50; 5, preparation after
Sephacryl S-200. Proteins were stained with Coomassie Brilliant
Blue R-250. Ten micrograms of protein was put on in all lanes.
cleavage enzyme from the corresponding catecholic
compounds, followed by measurement of the hydro-
lase reaction in a single tube. Depletion of the sub-
strates depended on the amount of CarC, and the
hydrolytic activity of CarC decreased in the substrate
EŠects of pH and temperature for activity and sta-
bility
The optimum pH for the enzyme activity was at
7.0 to 7.5. Hydrolase activity was relatively high
when the sodium citrate buŠer was used (data not
order of 6-(2
?-aminophenyl)-HODA (2.44 U mg)À
W
6-phenyl-HODA (1.99 U mg)
À
6-(2
?-hydroxy-
W
phenyl)-HODA (1.05 U mg). The activity for 6-(2
?-
W
hydroxyphenyl)-HODA was much higher than that
shown). CarC retained about 75
10-min incubation at 55 C or less, although incuba-
tion at 65 C inactivated the enzyme.
z activity after a
for 6-methyl-HODA and HODA.
9
To conˆrm whether the hydrolysis was actually
caused by CarC, we analyzed the products of the
enzymatic and nonenzymatic conversions of these 6-
9
Steady-state kinetic analysis
(2?-substituted phenyl)-HODAs. From the reaction
CarC obeyed Michaelis-Menten kinetics for 6-
phenyl-HODA, 6-methyl-HODA, and HODA. CarC
mixtures with CarB, the respective meta-cleavage
compounds were detected (Table 2). The cyclization
had only 0.38
HODA and HODA, respectively, with that toward 6-
phenyl-HODA taken to be 100 , indicating that
CarC was speciˆc for 6-phenyl-HODA. The Km of
CarC for 6-phenyl-HODA was 2.51 . The catalytic
m, were
z
and 0.54
z
activity toward 6-methyl-
product of 6-(2?
-hydroxyphenyl)-HODA40) was de-
tected. When CarC was added, anthranilate and
salycilate, which are the hydrolysis products of 6-(2?-
z
aminophenyl)-HODA and 6-(2?-hydroxyphenyl)-
HODA, respectively, were detected.
m
M
activity, kcat, and speciˆcity constant, kcat K
W
5
2.14 s„1 and 8.5 10 „1 s„1, respectively. We could
not measure the Km of CarC for other substrates be-
cause of its low activity.
M
Theoretical calculations
The physical features of 2?-substitution in the
phenyl moiety might aŠect the substrate speciˆcity of
CarC. In C-C bond hydrolysis, an nucleophilic at-
tack of water catalyzed by active site serine has been
proposed to occur.41) The lowest unoccupied molecu-
lar orbital (LUMO) could act as an electron acceptor,
×
Characterization of 6-(2
HODAs
The absorption spectra of the solutions of 6-(2
?-substituted phenyl)-
?-

C-C Bond Hydrolase from a Carbazole Degrader
41
Table 2. Identiˆcation of Transformation Products of 2,3-Dihydroxybiphenyl and Its Derivatives by CarB and CarC
GC-MS dataa
Enzyme
used
Substrate
Retention
time Principal ions and relative abundance (
(min)
Identiˆcation
z
base peak)
2,3-Dihydroxybiphenyl
CarB
CarB+CarC
-Aminobiphenyl-2,3-diol CarB
CarB+CarC
12.0 347(M-15+, trace), 257(2), 245(28), 147(16), 73(100) 6-Phenyl-HODAd
4.6 194(M+, 6), 179(100), 135(89), 105(87), 77(69)
12.6 377(M+, 3), 362(3), 260(26), 147(23), 73(100)
6.7b 209 (M+, 34), 194(76), 176(27), 150(54), 120(100)
7.8c 281(M+, 1), 266(38), 218(12), 147(18), 120(100)
12.6 377(M+, 3), 362(2), 260(21), 147(25), 73(100)
11.6 378(M+, 6), 293(10), 261(81), 193(20), 83(100)
Benzoatee
6-(2
-Aminophenyl)-HODAd
Anthranilateb
2?
?
,
e
e
Anthranilatec,
6-(2
?
-Aminophenyl)-HODAd
2,2?,3-Trihydroxybiphenyl CarB
3-(Chroman-4-on-2-
yl)pyruvatef
12.7 435(M-15+, 4), 407(3), 333(100), 257(22), 73(81)
7.0 267(M-15+, 20), 209(6), 193(2), 149(8), 73(100)
11.6 378(M+, 5), 293(6), 261(92), 193(48), 83(100)
6-(2?
-Hydroxyphenyl)-HODAf
CarB+CarC
Salycilatee
3-(Chroman-4-on-2-
yl)pyruvatef
12.7 435(M-15+, 4), 407(2), 333(100), 257(20), 147(44)
6-(2?
-Hydroxyphenyl)-HODAf
a
b
c
d
e
f
Results of GC-MS of trimethylsilyl derivatives.
Only the carboxyl group of anthranilate was trimethylsilylated.
Both the carboxyl and amino groups of anthranilate were trimethylsilylated.
The tentative identiˆcation was based on the results of GC-MS for the trimethylsilyl derivative of 6-(2
The identiˆcation was based on comparison with the results of GC-MS of authentic samples.
The identiˆcation was based on comparison with the results of Kohler et al
?
-hydroxyphenyl)-HODA.40)
40)
.
and the LUMO energy is equivalent to the electron
a‹nity of the molecule, according to Koopmans the-
orem.42) On the basis of frontier orbital theory,43)
when the energy diŠerence of the highest occupied
molecular orbital of a nucleophile and the LUMO of
an electrophile is small, the reactivity of nucleophilic
attack is high. We calculated the LUMO energies of
the substrates to evaluate the reactivity of nucleophil-
ic attack by water. The calculated LUMO energy was
tion of the oligomeric structure.
The optimum pH of meta-cleavage compound
hydrolases from Rhodococcus sp. strain RHA1 has
been reported to be 6.0 and 6.5,35) but the optimum
pH of hydrolases from pseudomonads has been
reported to be 7.4,47) 8.6,48) and 7.0–7.5.49) The opti-
mum pH of MhpC from E. coli has been reported to
be 8.5–9.0.50) The optimum pH for CarC, 7.0–7.5,
was similar to that for other meta-cleavage com-
pound hydrolases originating from pseudomonads.
lowest for 6-(2
higher for 6-phenyl-HODA („0.718), and highest
for 6-(2 -hydroxyphenyl)-HODA („0.671). This
?-aminophenyl)-HODA („0.754),
Incubation at 65
enzyme retained about 75
incubation at 55 C. At 80
9
9
C rapidly inactivated CarC, but the
activity after 10 min of
C or less, hydrolase activi-
?
z
predicted reactivity was in agreement with the ex-
perimental results.
9
ty was higher when the reaction temperature was in-
creased, and we could not detect a temperature for
maximum hydrolase activity (data not shown). Con-
sidering that the optimum temperatures for EtbD1
and BphD from Rhodococcus sp. strain RHA1 are
Discussion
The relative molecular weight of the native CarC
was estimated by chromatography on a Sephacryl
S-200 column, to be 70,000. CarC exhibited a
single band on SDS-PAGE, which corresponded to
31,000 Da, suggesting that the CarC enzyme is com-
posed of two identical subunits. Variation in the
oligomeric structures (monomer to homooctamer)
has been reported for meta-cleavage compound
hydrolases, and the oligomeric structure seems not to
be correlated with the substrate speciˆcity.18,35)
However, there may be an interface structure that
allows for speciˆc oligomeric structures of enzymes.
To date, the three-dimensional structures of BphD
(a homooctamer) from Rhodococcus sp. strain
RHA135,44) and CumD (a homodimer) from P.
‰uorescens strain IP0145,46) has been identiˆed. Com-
parison between the interface structures among the
hydrolases will provide information on the construc-
659and 459
C, respectively,35) CarC seems to be a
thermostable. It has been predicted that the car gene
cluster was constructed from several ancestral genes
through gene rearrangement,9) and we cannot clarify
the origin of the carC gene on the basis of the infor-
mation we have about the genetic structure of this
gene cluster. However, the characteristics of CarC
might re‰ect conditions in the cytosol of the bacteri-
um originally having the carC gene.
Almost all of the hydrolases involved in degrada-
tion of monocyclic aromatic compounds has been
classiˆed into group III. Phylogenetic analysis
showed close relationships with group III hydrolases
of CarCs of strain CA10 and Sphingomonas sp.
strain GTIN11,23) and DxnB of S. wittichii strain
RW1.24,25) Like DxnB of strain RW124,25) and other
hydrolases found in bacteria that degrade biphenyl

42
H. NOJIRI et al.
and polychlorinated biphenyl,18,35) CarC showed a
strict preference for 6-phenyl-HODA. Accordingly,
the substrate speciˆcity was not in accordance with
the results of phylogenetic analysis, indicating the
divergence of the structures and functions of the C-C
bond hydrolases involved in aromatic compound
degradation. Examination of the structure of the 6-
phenyl-HODA hydrolase, BphD, of Rhodococcus
sp. strain RHA1, showed that the subunit protein can
be divided into core and lid domains, and that the
substrate binding pocket was between the two
domains.44) The substrate binding pocket of the
BphD enzyme is composed of a hydrophilic P (prox-
imal) part and a hydrophobic D (distal) part. The
amino acid residues on the surface of the D part were
not well conserved among similar enzymes, suggest-
ing that the diŠerence in this part contributes to the
substrate speciˆcities of meta-cleavage compound
hydrolases. Part of the surface of the D part was
composed of the amino acid residues on the lid
domain.44) Amino acid sequence alignment showed
that the amino acid residues in the lid domains of
group I or III hydrolases were well conserved within
their groups; similarity was not found in the regions
between groups (not shown). In contrast, many ami-
no acid residues of the core domain were conserved
in almost all of the meta-cleavage compound hydro-
lases tested. The phylogeny observed in Fig. 1 prob-
ably originated in the main from divergence in the
amino acid sequence of the lid domain. If so, the
three-dimensional structure of the lid domain is
diverse, and CarC has a structure similar to the struc-
tures of the hydrolases of monocyclic compound
degradation pathways. However, the CarC substrate
speciˆcity, which is unusual for group III hydrolases,
indicates that there is some diŠerence in the D part of
the substrate binding pocket of CarC with other
group III hydrolases involved in this kind of degrada-
tion. Recently, the three-dimensional structure of
CumD, a hydrolase of the isopropylbenzene degrada-
tion pathway in P. ‰uorescens strain IP01 and classi-
ˆed into group III, has been identiˆed as a complex
form with a hydrolysis product.46) For comparison,
analysis of the three-dimensional structure of CarC is
in progress.
zyme, and subsequently monitored substrate deple-
tion by CarC. The results showed that CarC catalyze
the hydrolysis of the meta-cleavage compounds in-
volved in the degradation pathways of both carba-
zole and dibenzofuran. Compared with the eŠect of
the volume of the C6 side chain, the eŠect of the
presence of the amino group and hydroxyl group at
the 2?-position of the phenyl moiety of substrate on
the hydrolase activity was found to be small.
However, as shown above, the CarC enzyme cata-
lyzed the hydrolysis of 6-(2
more eŠectively than that of either 6-phenyl-HODA
or 6-(2 -hydroxyphenyl)-HODA. CarC hydrolyzes 6-
phenyl-HODA more than 6-(2 -hydroxyphenyl)-
?-aminophenyl)-HODA
?
?
HODA. That the car gene cluster of strain CA10 was
induced in cells cultivated on carbazole or anthrani-
late (Urata et al., unpublished) indicates that at least
a part of the car gene cluster is originally related to
nitrogen metabolism. This possibility is in agreement
with 6-(2?-aminophenyl)-HODA being a substrate
for CarC of strain CA10. The pattern of substrate
speciˆcity was in accordance with theoretical calcula-
tions based on the LUMO energy. However, there
may be various structures with local energy mini-
mum, because part of the carbon chain is ‰exible. In
addition, the eŠects of interaction with CarC would
be large. Therefore, after analyzing the structure of
CarC, it is expected that more exact analysis can be
done on the state of a substrate-enzyme complex.
Compared with the half-life of 6-phenyl-HODA
(58 h),19) 6-(2
?-aminophenyl)-HODA and 6-(2?-
hydroxyphenyl)-HODA were unstable under the
conditions used (6.1 and 7.3 min, respectively). 6-
Phenyl-HODA is spontaneously converted to
acetophenone.19) The hour-scale half-life of 6-phenyl-
HODA possibly originated from this spontaneous
transformation. On the other hand, intermolecular
cyclizations by the Michael-type addition of amino
and hydroxyl groups at the 2?-position of meta-
cleavage compounds in the carbazole and dibenzofu-
ran degradation pathways to the double bond of the
aliphatic side chain occurred mainly as a spontaneous
transformation.40,53,54) This reaction may cause the
meta-cleavage compounds in carbazole and diben-
zofuran degradation pathways to be unstable. When
strain CA10 is cultivated on carbazole as the sole
carbon, nitrogen, and energy source, anthranilate
accumulates in the culture supernatant.6) This ˆnding
indicates that angular dioxygenase, meta-cleavage
enzyme, and hydrolase can catalyze the conversion
of carbazole to anthranilate more eŠectively than
the subsequent conversion of anthranilate. This
phenomenon might indicate the presence of an
unknown mechanism to allow the eŠective sequential
metabolism of aromatic compounds to avoid the
spontaneous degradation of the unstable intermedi-
Several meta-cleavage compound hydrolases in-
volved in the dibenzofuran and carbazole degrada-
tion pathways have been characterized.8,24,51) In those
studies, 6-phenyl-HODA was used as a model sub-
strate to discover the enzymatic features of the
hydrolases, because of the unstability of 6-(2?-sub-
stituted phenyl)-HODAs.52) We compared the hydro-
lase activity of CarC for the primary substrate, 6-
(2?-aminophenyl)-HODA, with that for 6-(2?-
hydroxyphenyl)-HODA and 6-phenyl-HODA. To
overcome bias arising from the short half-lives of the
substrates, we used a combination assay in which the
substrate was produced by the meta-cleavage en-
ate, 6-(2?-aminophenyl)-HODA.

C-C Bond Hydrolase from a Carbazole Degrader
43
Omori, T., Diverse oxygenations catalyzed by carba-
zole 1,9a-dioxygenase from Pseudomonas sp. strain
CA10. J. Bacteriol., 181, 3105–3113 (1999).
Acknowledgments
Part of this work was supported by the Program
for Promotion of Basic Research Activity for In-
novative Bioscience (PROBRAIN) in Japan.
14) Habe, H., Chung, J.-S., Lee, J.-H., Kasuga, K.,
Yoshida, T., Nojiri, H., and Omori, T., Degradation
of chlorinated dibenzofurans and dibenzo-
p-dioxins
by two types of bacteria having angular dioxygenase
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