(Received for publication, February 26, 1997, and in revised form, April 4, 1997)
From the Division of Science of Biological Resources, 2-Aminophenol 1,6-dioxygenase was purified from
the cell extracts of Pseudomonas sp. AP-3 grown on
2-aminophenol. The product from 2-aminophenol by catalysis of the
purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas
chromatographic and mass spectrometric analyses. The molecular mass of
the native enzyme was 140 kDa based on gel filtration. It was
dissociated into molecular mass subunits of 32 ( Dioxygenases catalyzing the fission of benzene rings are key
enzymes in the metabolic pathways of aromatic compounds by
microorganisms. Most of these kinds of previously reported dioxygenases
attack monocyclic aromatic compounds with two adjacent hydroxyl groups such as catechol and protocatechuic acid and open the benzene rings
through the intradiol or extradiol fission reaction (1, 2). However,
some bacteria have been reported to synthesize dioxygenases that cleave
the benzene rings of hydroquinone (3-5) and gentisic acid (6, 7).
In our investigations on the microbial metabolism of anilines, we
isolated several microorganisms capable of growing on 2-aminophenol as
the sole carbon, nitrogen, and energy source. When one isolate, Pseudomonas sp. AP-3, grows with this substrate, it
synthesizes an enzyme acting on 2-aminophenol. This enzyme was
partially purified with a 103-fold increase in the specific activity
from its cell extracts. We proposed that the enzyme is a dioxygenase
catalyzing the ring fission of 2-aminophenol with the consumption of 1 mol of O2 per mol of substrate (8).
Our aim was to advance the purification of 2-aminophenol
1,6-dioxygenase from Pseudomonas sp. AP-3 and elucidate the
molecular and catalytic properties of the purified enzyme. Because the
product from 2-aminophenol by catalysis of the enzyme is rapidly and
nonenzymatically converted into picolinate (8, 9), the real product has
remained unverified. Furthermore, we attempted the cloning and
sequencing of the gene of the dioxygenase, which would determine the
category of this enzyme in the dioxygenase groups.
Recently, Lendenmann and Spain (10) reported the purification and
characterization of the 2-aminophenol 1,6-dioxygenase from
nitrobenzene-degrading Pseudomonas pseudoalcaligenes JS45, although they did not refer to the cloning and sequencing of its gene.
In this report, the comparison of the dioxygenases from the two
pseudomonads growing on 2-aminophenol or nitrobenzene is also
described.
The chemicals used in this study and their
sources are as follows. Polypepton, 2-aminophenol, catechol,
4-methylcatechol, methyl chlorocarbonate, and
N,O-bis(trimethylsilyl)trifluoroacetamide were
from Wako Pure Chemicals, Osaka; 2-amino-p-cresol,
6-amino-m-cresol, 2-amino-4-chlorophenol, 3-methylcatechol,
3-chlorocatechol, 4-chlorocatechol, and 3-fluorocatechol were from
Tokyo Kasei, Tokyo; pentafluorophenylhydrazine, 2-amino-m-cresol, and 2-amino-4,5-dimethylphenol were
from Aldrich; DE52 cellulose was from Whatman; DEAE-Cellulofine A-500
was from Seikagku Co., Tokyo; and restriction endonucleases were from
Takara Shuzo, Otsu.
Pseudomonas sp. AP-3 was used
throughout this study as a producer of 2-aminophenol 1,6-dioxygenase
and a donor of its gene. Escherichia coli JM109 and E. coli P2392 were used as hosts for the recombinant plasmids and
bacteriophages, respectively. A lambda FIX II/XhoI partial
fill-in vector (Stratagene, La Jolla) was used for the construction of
a gene library. pGEM-T (Promega, Madison) and pBluescript II SK(+)
vectors (Stratagene) were used for cloning of the PCR (polymerase chain
reaction) products and subcloning of the DNA fragments,
respectively.
Pseudomonas sp.
AP-3 was cultured in the 2-aminophenol medium (8) containing 0.12%
(w/v) of the substrate and supplemented with 1% (w/v) Polypepton.
Pseudomonas sp. AP-3 used for isolating its total DNA and
E. coli strains were cultured in Luria broth (11) with
shaking at 30 and 37 °C, respectively.
The activity of 2-aminophenol 1,6-dioxygenase
was measured by monitoring the decrease in absorbance at 282 nm
according to a previous paper (8). The activities for the 2-aminophenol analogs were measured by scanning changes in the absorbance of each
reaction mixture, because all substrates tested had absorption bands in
the UV range. Molar extinction coefficients of the substrates attacked
by the enzyme were determined in this study as follows: 3100 at 287 nm
for 2-amino-p-cresol, 2700 at 287 nm for
6-amino-m-cresol, 3100 at 291 nm for 2-amino-4-chlorophenol,
2100 at 279 for 2-amino-m-cresol, and 2600 at 289 nm for
2-amino-4,5-dimethylphenol. For catechol, catechol 1,2-dioxygenase (12)
and catechol 2,3-dioxygenase (13) activities were assayed. Protein
concentrations were measured by the method of Lowry et
al. (14).
All
operations for enzyme purification were done at 0-4 °C, and
centrifugation was carried out at 20,000 × g for 10 min. The frozen cells (37 g, wet weight) of Pseudomonas sp.
AP-3 were used for the purification. The preparation of the cell
extracts (step 1, fraction 1), streptomycin sulfate treatment (step 2, fraction 2), and ammonium sulfate fractionation (step 3, fraction 3)
were essentially carried out by the same methods as described
previously (8).
After the protein
concentration of fraction 3 was adjusted to 7 mg ml Fraction 4 was
applied to a column (2.2 × 27 cm) of DE52 cellulose equilibrated
with buffer A. Proteins were eluted with a linear gradient (0 to 0.4 M) of NaCl in 1.4 liters of buffer A, and then the protein
concentrations and 2-aminophenol 1,6-dioxygenase activities were
assayed. Fractions with the specific activity higher than 2.7 units
mg Fraction 5 was dialyzed against buffer A. The dialyzed solution was applied to a
column (2 × 26 cm) of DEAE-Cellulofine A-500 equilibrated with
buffer A. The enzyme was eluted with a linear gradient (0 to 0.35 M) of NaCl in 1.4 liters of buffer A. The enzyme in each
fraction was tested by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)1 (15). Fractions
showing two distinct protein bands ( The molecular mass of the
native enzyme was measured by the two methods of gel filtration and
PAGE (16). Those of the enzyme subunits were measured by SDS-PAGE
(15).
The reaction mixture consisted of 5 mM
2-aminophenol, 12 ml; 2-aminophenol 1,6-dioxygenase, 10 mg in 10 ml of
buffer A; and 100 mM sodium-potassium phosphate buffer (pH
7.5), 330 ml. The reaction started at 24 °C by adding the enzyme
solution with shaking. After 1 min, 2.4 ml of methyl chlorocarbonate
was added to the mixture, and the reaction was continued for 10 min
(17). The reaction mixture was then concentrated to 100 ml with a
rotary evaporator. After the pH of the concentrated solution was
adjusted to pH 3.0 with 3 N HCl, the solution was extracted
with ethyl acetate. The upper layer was then evaporated to dryness. The
residues were dissolved in 10 ml of methanol and reacted with 34 mg of pentafluorophenylhydrazine for 1 h at room temperature (18). The
hydrazone derivative produced was trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide and
analyzed using a Hitachi M-2500 mass spectrometer at an ionization
potential of 70 eV, coupled to a Hitachi G-3000 gas chromatograph. A
TC-1 fused silica capillary column (GL Sciences, Tokyo) was used.
Iron in the enzyme was determined using
o-phenanthroline after it was reduced to Fe2+
with hydroxylamine·HCl (19).
SDS-PAGE was employed to dissociate the purified enzyme
into the Chromosomal DNA of Pseudomonas sp. AP-3 was
prepared according to the protocol described by DiLella and Woo (21).
The purified DNA (30 µg) was partially digested with
Sau3AI, and DNA fragments larger than 23 kb were ligated
into a lambda FIX II/XhoI partial fill-in vector after
fill-in of the first two nucleotides of the Sau3AI-compatible site, according to the manufacturer's
instructions. Gigapack III gold (Stratagene) was used to package the
recombinant lambda phages. The infection of phage particles into
E. coli P2392 and recovery of the recombinant phage DNA from
the top agar were performed using the standard method (11). Subcloning
experiments were performed using conventional techniques (11).
On the basis of NH2-terminal amino acid
sequences of
Amplified DNA fragments
were ligated into a vector and partially sequenced to ensure that the
ligated fragments contained an amn gene. After sequencing,
the recombinant plasmid containing the amn gene was digested
with SacII and PstI. The inserted DNA fragments
were separated from the vector DNA using 1% agarose gel
electrophoresis, recovered from gel slices by GeneClean II (Bio 101, La
Jolla), and radiolabeled with [ Genomic DNA from Pseudomonas sp.
AP-3 was digested with various restriction endonucleases. The DNA
fragments were transferred onto a Hybond-N membrane (Amersham Corp.)
with a probe tech 2 (Oncor Inc., Gaithersburg) according to the
manufacturer's instructions after 1% agarose gel electrophoresis.
Plaque and Southern blot hybridizations for DNA were performed by the
standard methods (11). Autoradiograms were analyzed with a Fuji BAS2000
bio-image analyzer (Fuji Film, Tokyo) after 3-4 h of exposure at room
temperature.
A Qiagen plasmid mini kit (Qiagen, Hilden)
was used for preparing the double-stranded DNAs for sequencing.
Sequencing reactions were performed by using a dye terminator cycle
sequencing FS ready reaction kit or dye primer cycle sequencing FS
ready reaction kit (Applied Biosystems). Reaction mixtures were run on
a 373 DNA sequencer (Applied Biosystems).
Table
I shows a summary of a typical enzyme purification. The
specific activity of the final preparation of 2-aminophenol 1,6-dioxygenase was 4.8 units mg Table I.
Purification of 2-aminophenol 1,6-dioxygenase
Densitometric analyses of the bands revealed that the molar ratio of
the two subunits was one to one on the basis of these molecular sizes.
In addition, the molecular mass of the native enzyme was 140 kDa by gel
filtration. These findings indicate that the enzyme was made up of four
heterogeneous subunits with the structure of
The purified enzyme was stable in buffer A at 4 °C for a week
without any decrease in activity. However, it lost activity within
24 h in the absence of ethanol, dithiothreitol, and
L-ascorbate.
Fig. 2 shows the
mass spectra of the N-acylated and trimethylsilylated
hydrazone derivative of compound I. There was a molecular ion at
m/z 451 (M+), which agreed with the empirical
formula of
C17H18F5N3O4Si. Major fragment ions appeared at m/z 436 (M+-CH3), 419 (M+-OCH3-H), 404 (M+-OCH3-CH3-H), 391 (M+-COOCH3-H), 377 (M+-COOCH3-NH), 361 (M+-COOCH3-2CH3-H), 302 (M+-COOCH3-OSi(CH3)3-H),
274(M+-COOCH3-H-COOSi(CH3)3),
255 (M+-C6F5-NH-N), 242 (M+-C6F5-NH-N-CH), 229 (M+-C6F5-NH-N-2CH), 195 ([C6F5N2]+), and 73 ([Si(CH3)3]+). These data showed
that compound I was 2-aminomuconic 6-semialdehyde.
The enzyme did not have any absorption
band in the visible range. However, it had an absorption peak at 280 nm
and a small shoulder at 287-290 nm: E1%
=13.7 × 104 at 280 nm. The
E280/E260 ratio of the
enzyme was 1.8.
The enzyme contained 0.98 mol of
Fe2+ per mol of protein on the basis of the molecular mass
of 140 kDa.
The substrate specificity of
2-aminophenol 1,6-dioxygenase was examined with 39 aromatic compounds
consisting of catechol, phenol, and aniline compounds (Table
II). Besides 2-aminophenol, the enzyme was active toward
2-amino-p-cresol, 6-amino-m-cresol, 2-amino-m-cresol, 2-amino-4,5-dimethylphenol,
2-amino-4-chlorophenol, and catechol. However, it did not act on
2-aminophenol analogs that were substituted by a carboxyl or nitro
group at the 3-, 4-, and 5-positions. In addition, catechol compounds
except catechol were not substrates of the enzyme.
Table II.
Substrate specificity of 2-aminophenol 1,6-dioxygenase
Department
of Biofunctional Chemistry,
-subunit) and 40 kDa
(
-subunit) by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, indicating that the dioxygenase was a heterotetramer
of
2
2. The genes coding for the
- and
-subunits of the enzyme were cloned and sequenced. Open reading
frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively. The amino acid sequences predicted from the open reading frames of amnA and
amnB corresponded to the NH2-terminal amino
acid sequences of the
-subunit (AmnA) and
-subunit (AmnB),
respectively. The deduced amino acid sequences of AmnB showed
identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are
homoprotocatechuate 2,3-dioxygenases from Escherichia coli
W and C, respectively, belonging to class III in the extradiol
dioxygenases. On the other hand, AmnA had identity (23.3%) with only
AmnB among the enzymes examined.
Chemicals
1 by
adding buffer A (20 mM Tris-HCl (pH 8.0) containing 10%
(v/v) ethanol, 1 mM dithiothreitol, and 0.5 mM
L-ascorbate), acetone was added to the diluted solution to
give 55% (v/v). The precipitate was removed by centrifugation and then
acetone was added to the supernatant to give 65% (v/v). The
precipitate was obtained by centrifugation and then dissolved in buffer
A. The enzyme solution was dialyzed against buffer A (fraction 4).
1 were pooled (fraction 5).
- and
-subunits) on the gel
were pooled (fraction 6).
- and
-subunits. The separated subunits were
electroblotted using the method of Matsudaira (20) and then
sequenced.
- and
-subunits of 2-aminophenol 1,6-dioxygenase,
the four primers
1,
2,
1, and
2 were synthesized (Fig. 3).
2 and
2 had complementary sequences to
1 and
1,
respectively. For amplifying fragments containing an 2-aminophenol
1,6-dioxygenase gene (amn gene) by PCR, Takara Ex
Taq was used with various combinations of the primers. The
cycling of PCR was performed at 94 °C (1 min), 51 °C (2 min), and
72 °C (2 min) for 25 cycles.
Fig. 3.
NH2-terminal amino acid sequences
of the - and
-subunits and their corresponding primer
sequences. The primers of
1 and
1 were synthesized on the
basis of coding strands, and the
2 and
2 primers were anticoding
strands. I, Y, S, R, and W in primer sequences
indicate inosine, CT, GC, AG, and AT, respectively.
[View Larger Version of this Image (18K GIF file)]
-32P]dCTP (Amersham
Corp., Buckinghamshire) and a random primer DNA labeling kit version 2 (Takara).
Purification of Enzyme and Its Molecular Properties
1 with an overall
recovery of 36%. A 120-fold increase in the specific activity was
observed at the final step of the purification procedure. The final
enzyme preparation showed one major protein band and two indistinct
bands on a polyacrylamide gel without SDS. The molecular mass of the
major band was 146 kDa on the gel (16). Those of other two bands were
lower than 85 kDa. However, on a SDS-polyacrylamide gel, the final
preparation showed two distinct protein bands with molecular masses of
32 kDa (
-subunit) and 40 kDa (
-subunit) (Fig.
1).
Fraction
Volume
Total activity
Total protein
Specific
activity
Recovery
ml
units
mg
units/mg
%
1. Cell extract
460
160
4300
0.04
100
2. Streptomycin sulfate
463
150
4300
0.03
94
3. Ammonium sulfate
97
230
1500
0.15
140
4. Acetone
58
260
500
0.52
160
5. DE52
49
74
26
2.8
46
6. DEAE-cellulofine
25
58
12
4.8
36
Fig. 1.
SDS-PAGE of the purified 2-aminophenol
1,6-dioxygenase. a, SDS-PAGE. The enzyme (10 µg) was run
on a 7.5% gel containing 0.1% (w/v) SDS (15). The gel was stained
with 0.25% (w/v) Coomassie Brilliant Blue R-250 in a solvent of
ethanol/acetic acid/H2O (9:2:9). b,
densitometric analyses of the protein bands. An Atto AE-6920 densitograph (Atto, Tokyo) was used.
[View Larger Version of this Image (34K GIF file)]
2
2.
Fig. 2.
Mass spectrum of a derivative of the reaction
product.
[View Larger Version of this Image (23K GIF file)]
Substrate
Relative
activity
Inhibition of 2-aminophenol turnover
Ki (µM)/type of inhibition
%
2-Aminophenol
100
2-Amino-p-cresol
10.0
+a
6-Amino-m-cresol
19.1
+a
2-Amino-m-cresol
4.4
+a
2-Amino-4,5-dimethylphenol
1.3
+a
2-Amino-4-chlorophenol
6.8
+a
Amidol
0
±b
4-Aminoresorcinol
0
9.1 /Noncompetitive
3-Hydroxyanthralinic acid
0
±b
Catechol
2
10.4 /Noncompetitive
3-Methylcatechol
0
6.8 /Noncompetitive
4-Methylcatechol
0
9.5 /Uncompetitive
3-Fluorocatechol
0
5.4 /Noncompetitive
3-Chlorocatechol
0
6.7 /Competitive
4-Chlorocatechol
0
10.4 /Competitive
Pyrogaroll
0
8.4 /Noncompetitive
1,2,4-Trihydroxybenzene
0
63.0 /Noncompetitive
a
The numerical values were not calculated, because
maximal absorption wavelengths of 2-aminophenol and its analogs used
for the measurement of the enzyme activity were overlapped.
b
Slightly inhibited.
The Km and
Vmax values for 2-aminophenol of 2-aminophenol
1,6-dioxygenase were 46.7 µM and 0.10 µM
s1 mg
1, respectively. The enzyme for
2-aminophenol as the substrate was inhibited by the catechols and
4-aminoresorcinol listed in Table II. In addition, the 2-aminophenol
analogs that could be degraded by the enzyme also inhibited it from the
action on 2-aminophenol, although their types of inhibition were
unmeasured. The activity of the enzyme for 2-aminophenol was not
affected by the 2-aminophenol analogs such as phenols and anilines that
were not substrates of this enzyme.
The effects of metal salts, chelating and sulfhydryl agents on the enzyme activity were tested using 2-aminophenol as the substrate (Table III). Among the metal ions tested, the enzyme was strongly inhibited by CuSO4, FeCl3, K3Fe(CN)6, AgNO3, HgCl2, or MnCl2. FeSO4 and MgSO4 did not inhibit the enzyme very much, and FeSO4(NH4)2SO4 slightly increased the activity for 2-aminophenol. Chelating agents and NaN3, completely repressed the enzyme activity.
|
The amino acid
sequences of 30 and 20 residues of the - and
-subunits,
respectively, of the enzyme were determined. On the basis of the two
sequences, the four primers
1,
1,
2, and
2 were synthesized
(Fig. 3).
When the 1 and
2
primers and DNA purified from Pseudomonas sp. AP-3 as a
template were incubated, a 1-kb DNA fragment was amplified. The
sequencing of both termini of the amplified fragment showed that this
fragment encoded a large portion of the
-subunit and an
NH2-terminal region of the
-subunit and that the
-subunit gene was located upstream of the
-subunit gene. The
sequenced fragment was labeled with [
-32P]dCTP and was
used as a probe for Southern hybridization. This DNA probe hybridized
to the DNA fragments from the AP-3 strain digested with several
restriction endonucleases (Fig. 4). These results showed
that the PCR product was amplified on the basis of the DNA sequence
from the AP-3 strain. The appearance of two positive bands for the
KpnI- (lane 7) and SacI- (lane
8) digested DNAs suggests that the PCR product contained
recognition sites for KpnI and SacI in its
sequence.
Cloning of amn Gene
The genomic library of
Pseudomonas sp. AP-3 was constructed in a lambda FIXII phage
vector. The hybridization to about 3000 plaques was performed using the
probe for the amn gene mentioned above. After screening
twice, we obtained five positive clones, p3-1, p4-3, p5-2, p12-2, and
p12-7. The DNAs purified from these phage clones were digested with
ApaI, BanIII, EcoRI, EcoRV,
KpnI, and SacI. Agarose gel electrophoretic
analyses revealed that restriction fragments obtained from these DNAs
contained several fragments with the same size as the positive bands
detected in the Southern hybridization (Fig. 4). The p4-3 DNA was
selected for subcloning of the amn gene, because it was
recovered with the greatest yield of the obtained fragments.
SacI (1.7-kb) and EcoRI (1.4-kb) fragments encoding whole - and
-subunit genes, respectively, were separated from each other and ligated into a pBluescript II SK(+) vector. The
obtained pS1 and pE1 plasmids carried 1.7-kb SacI and 1.4-kb EcoRI fragments, respectively.
The DNA fragments inserted into pS1 and pE1 were
sequenced. Fig. 5a shows that 884 base pairs
of the two fragments overlapped. Two open reading frames containing
each primer sequence used in the PCR were found in the connected
sequence and were designated as amnB for the first open
reading frame and amnA for the second one. Fig.
5b shows a DNA sequence connected with pE1 and pS1, and
amino acid sequences deduced from amnB and amnA.
The amnB encoded 305 amino acid residues. The deduced amino
acid sequences from positions 2 to 31 completely agreed with the
NH2-terminal amino acid sequences of the -subunit
determined from the purified enzyme. The initiation codon (ATG) of
amnA started 32 base pairs downstream from the termination
codon (TAA) of amnB. AmnA was made up of 271 amino acid
residues and agreed with the NH2-terminal amino acid
sequences of the
-subunit from positions 2 to 21.
The 2-aminophenol 1,6-dioxygenase from Pseudomonas sp.
AP-3 growing on 2-aminophenol was purified with a 120-fold increase in
the specific activity. Although the final preparations of the enzyme
did not show one band on PAGE under nondenaturing conditions, these
showed distinct two protein bands on the SDS-PAGE gel (Fig. 1). The two
components were made up of an equal molar ratio on the basis of the
molecular masses of 32 (-subunit) and 40 kDa (
-subunit). In
addition, each protein band electroblotted onto the transfer membrane
for sequencing exhibited only one amino acid residue at the
NH2 terminus. Therefore, we consider that the final
preparations of 2-aminophenol 1,6-dioxygenase were homogeneous and
dissociated into several components by native PAGE. It was also
observed that the dioxygenase from the nitrobenzene-degrading P. pseudoalcaligenes JS45 shows one main band and two diffuse bands
on a PAGE gel at room temperature, although it exhibited a single band
at 10 °C (10).
Since the product from 2-aminophenol by the reaction of 2-aminophenol 1,6-dioxygenase was labile, the direct evidence for the chemical structure of the product has not been reported. In this study, after the amino group of the product was acylated with methyl chlorocarbonate, its aldehyde group was modified with pentafluorophenylhydrazine to prevent its cyclization to picolinate. Based on the gas chromatography-mass spectrometry analyses of the modified compound, we could prove that the real product from 2-aminophenol was 2-aminomuconic 6-semialdehyde. On the basis of the fact together with our previous report (8), it was concluded that the dioxygenase catalyzes the ring fission between the 1- and 6-positions of 2-aminophenol with the consumption of 1 mol of O2 per mol of substrate.
The 2-aminophenol 1,6-dioxygenase from P. pseudoalcaligenes
JS45 was described to be stable in 50 mM MOPS (pH 7.3)
containing 10% (v/v) glycerol (10). However, it is inactivated in the
buffer containing 10% (v/v) ethanol. When it was chromatographed on
MonoQ (Pharmacia Biotech Inc.) using the buffer without
Fe2+ or cysteine, the activity of this enzyme is completely
lost. The enzyme contains 2.2 mol of Fe2+ per mol of
enzyme. On the other hand, our dioxygenase from Pseudomonas sp. AP-3 was stable in buffer A containing 10% (v/v) ethanol and was
not stabilized in the presence of 10% (v/v) glycerol. The enzyme was
inactivated in the presence of Fe2+ or cysteine, or both.
In addition, the enzyme had 0.98 mol of Fe2+ per mol of
enzyme. Protocatechuate 4,5-dioxygenase was also reported to have
approximately 1 mol of Fe2+ per mol of enzyme, although the
enzyme is a heterotetramer of 2
2 (22).
These data showed that the two 2-aminophenol 1,6-dioxygenases are
distinctly different in stability against various factors and in
Fe2+ content, although these are essentially similar to
each other with respect to subunit structure, substrate specificity,
and inhibited properties.
We cloned the amnA and amnB genes encoding the
- and
-subunits, respectively, of the 2-aminophenol
1,6-dioxygenase from Pseudomonas sp. AP-3 and determined
their nucleotide sequences. This is the first report describing the
cloning and sequencing of the 2-aminophenol 1,6-dioxygenase genes.
Lendenmann and Spain (10) reported a part of the
NH2-terminal amino acid sequences of the
- and
-subunits of the 2-aminophenol 1,6-dioxygenase from P. pseudoalcaligenes JS45. The sequence comparison of AmnA (
-subunit) from the AP-3 strain with the
-subunit from the JS45 strain revealed that they contained 25 identical amino acid residues of
the 30 comparable residues (83% identity); AmnB (
-subunit) from the
AP-3 strain and the
-subunit from the JS45 strain showed 21 identical amino acid residues of the 30 comparable residues (70%
identity). The comparison of the amino acid sequences of AmnB with
those deduced from DNA data bases revealed that it had amino acid
sequence identities of 25.5 and 24.4% with HpaD (23) and HpcB (24),
respectively, which are homoprotocatechuate 2,3-dioxygenases from
E. coli strains (Fig. 6). However, AmnB did
not show any identity with other extradiol dioxygenases such as XylE
(catechol 2,3-dioxygenase) (25) encoded on the TOL plasmid and several 2,3-dihydroxybiphenyl 1,2-dioxygenases (26, 27).
HpaD and HpcB belong to class III in the extradiol dioxygenases proposed by Spence et al. (28). They described that the enzymes of class III possess an NH2-terminal domain containing the active center consisting of a Fe2+ cofactor and four histidine residues that are conserved in these enzymes (shaded area in Fig. 6). Multiple alignments of AmnB and the class III enzymes revealed that AmnB retains three histidine residues, His-14, His-63, and His-196, of the four histidine residues conserved in the class III enzymes; one residual histidine residue is replaced by an arginine residue at the corresponding position in AmnB (Fig. 6). At the same position in human 3-hydroxyanthranilic-acid dioxygenase belonging to class III, the histidine residue conserved in the class III enzymes was replaced by a threonine residue (28). In addition, we found the identical amino acid residues with those in the class III enzymes, which are located in the vicinity of the three conserved histidine residues. They were Pro-16, Glu-51, Leu-57, Ser-191, and Ser-195 in AmnB (Fig. 6). These results suggest that the amnB gene has evolved from a common ancestor gene for the class III enzymes and that amnB is classified into class III.
On the other hand, AmnA showed 23.3% identity with only AmnB among the enzymes examined (Fig. 6). The amino acid sequences common to the two proteins were dispersed in their sequences. This suggests that amnA and amnB would have diverged by the duplication of an ancestor gene. Multiple alignments of AmnA and the class III enzymes revealed that the three amino acid residues Pro-16, Val-147, and Ser-195 are conserved in every sequence of the enzymes listed in Fig. 6, although they are not catalytically active histidine residues (marked by ! in Fig. 6). The similarity of AmnA with AmnB and the existence of the conserved amino acid residues in AmnA may support the fact that AmnA also belongs to class III in extradiol dioxygenases. Because AmnA lacks the functional histidine residues conserved in other class III enzymes, it may be independent of the catalytic process of 2-aminophenol 1,6-dioxygenase and therefore play other roles, such as the stabilization of AmnB in the enzymatic reaction.
Among the class III enzymes whose amino acid sequences have been reported, only protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis SYK6 consists of two distinct subunits, LigA and LigB (29). However, it is noted that LigA, the small subunit of protocatechuate 4,5-dioxygenase, showed no identity with AmnA, the small subunit of 2-aminophenol 1,6-dioxygenase, although LigB aligned with AmnB. This fact suggests that LigA and AmnA are different in the process of molecular evolution and in the function of enzymatic catalysis.
Further efforts are now in progress to identify other genes responsible for the 2-aminophenol metabolism using the cloned DNA fragments from Pseudomonas sp. AP-3.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D89855[GenBank].