Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1

Article abstract of DOI:10.1023/A:1013126723719

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of 45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the K_m and V_max values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate. The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate 1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.

Full text of DOI:10.1023/A:1013126723719

Biodegradation 12: 179–188, 2001.
2001 Kluwer Academic Publishers. Printed in the Netherlands.
1
79
©
Purification and characterization of a salicylate hydroxylase involved in
-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and
phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1
1
1
2
2
3∗
3
N.V. Balashova , A. Stolz , H-J. Knackmuss , I.A. Kosheleva , A.V. Naumov & A.M.
3
Boronin
1
Pushchino State University, 142290, Pushchino, Moscow region, Russia; 2Institut für Mikrobiologie der
Universität Stuttgart, Allmandring 31,D-70569 Stuttgart, Germany; 3Laboratory of Plasmid Biology, Insti-
tute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino,
∗
Moscow region, Russia ( author for correspondence: tel: 7-095-956337499; fax: 7-095-9563370; e-mail:
kosheleva@ibpm.serpukhov.su
Accepted 2 February 2001
Key words: 1-Hydroxy-2-naphthoate, naphthalene, phenanthrene, Pseudomonas putida, salicylate hydroxylase
Abstract
1
-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxy-
genase which catalyzed the oxidation of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene was purified from
the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a
molecular weight of 45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed
the oxidation of salicylate and various substituted salicylates. The comparison of the Km and Vmax values for 1-
hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a
substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate.
The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-
monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of
phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate
1
-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are
sufficient to catalyse also the mineralization of phenanthrene.
Introduction
al. 1976) or via salicylate and catechol (Kiyohara &
Nagao 1978; Evans et al. 1965) (Figure 1).
The initial enzyme involved in the conversion of
-hydroxy-2-naphthoate to protocatechuate is a 1-
hydroxy-2-naphthoate 1,2-dioxygenase which cleaves
-hydroxy-2-naphthoate to 2-carboxybenzopyruvate.
This enzyme has been previously described and char-
acterized in Aeromonas sp. s45pl and Nocardioides sp.
Certain bacteria utilize polycyclic aromatic hydrocar-
1
bons (PAHs), including phenanthrene, as a sole source
of carbon and energy. The initial steps of phenanthrene
degradation lead to the formation of 1-hydroxy-2-
naphthoate (Kiyohara & Nagao 1978; Stringfellow
1
&
Aitken 1994; Evans et al. 1965; Kiyohara et al.
(
Kiyohara et al. 1976; Iwabuchi & Harayama 1998).
1
976) and might be catalysed by the same enzymes
The amino acid sequence of this enzyme differs from
those of other ring-cleaving dioxygenases that oxid-
ize double hydroxylated aromatic rings (Iwabuchi &
Harayama 1998).
which also convert naphthalene to salicylate (Menn
et al. 1993; Kiyohara et al. 1994). The 1-hydroxy-
2
-naphthoate which is formed from phenanthrene is
further metabolized by two different pathways, either
through o-phthalate and protocatechuate (Kiyohara et

1
80
Figure 1. Phenanthrene and naphthalene degradation pathways (Kiyohara & Nagao, 1978). (1) 1-Hydroxy-2-naphthoate hydroxylase, (2)
salicylate hydroxylase.
In contrast to the situation for the direct ring-fission
of 1-hydroxy-2-naphthoate, the enzyme which cata-
lysis the oxidative decarboxylation of 1-hydroxy-2-
naphthoate to 1,2-dihydroxynaphthalene has not been
characterized previously. To find out the enzyme
which is responsible for this reaction, we have chosen
to study the metabolism of 1-hydroxy-2-naphthoate
by the phenanthrene-degrading strain Pseudomonas
putida BS202-P1.
Methods
Media and growth conditions
Pseudomonas putida strain BS202-P1 was cultivated
◦
at 30 C on a rotary shaker (150 rpm) in 500-ml
Erlenmeyer flasks containing 200 ml of the mineral-
salt Evans medium (Evans et al. 1970). Naphthalene
and phenanthrene were added to liquid media as
−1
crystals to final concentrations of 1 and 0.2 g l
,
respectively. 1-Hydroxy-2-naphthoate and salicylate
were added as growth substrates to final concentra-

1
81
tions of 0.5 and 1 g l 1, respectively. Growth with
−
tion coefficient for 1-hydroxy-2-naphthoate at 340 nm
−1
−1
1
-hydroxy-2-naphthoate for enzyme purifications was
performed at 30 C in a “1601 Ultroferm” fermenter
7 l working volume, LKB, Sweden). The fermenter
was oxygenated by air (agitation at 250 rpm).
Cells used in induction experiments were grown in
liquid mineral salt medium containing 1 g l succin-
ate. In the early exponential growth phase salicylate
and 1-hydroxy-2-naphthoate were added at 0.1 g l
respectively, and the bacteria grown for another two
hours, before the cells were harvested.
was determined as 5.08 mM cm . Therefore, a
◦
−1
molar reaction coefficient at 340 nm of 11.3 mM
−1
(
cm was used to calculate the reaction of 1-hydroxy-
2-naphthoate and NADH to 1,2-dihydroxynaphthalene
and NAD.
−1
For the determination of the pH optimum of the
enzyme, the standard spectrophotometric assay was
used and the pH values of the phosphate buffers were
varied from 6.0 to 8.0.
−1
,
Protein was determined by the Bradford method
(
Bradford 1976) using bovine serum albumin as a
Preparation of cell extracts
standard.
◦
Cells were harvested by centrifugation at 4 C, washed
Protein purification
first with 0.5 M NaCl, then with 80 mM NaCl and
finally with 20 mM KH2PO4 buffer, pH 7.5. Approx-
imately 6 g (wet weight) of cell paste was suspended
in 10 ml of 20 mM KH2PO4 buffer, pH 7.5. Cells
were lysed using a IBPM-press (Russia) at 7 mPa and
All purification procedures were carried out by using
a FPLC system (UV-1 control unit, P-500 conduct-
ivity monitor, LCC-501 Plus controller, LKB REC
102 recorder, FRAC-100 collector, UV-1 optical unit,
Pharmacia). Crude cell extract was loaded onto a
Pharmacia Resource Q column (bed volume 6 ml,
16 × 30 mm). Proteins were eluted with a linear
KH PO (pH 7.5) gradient (20 mM to 200 mM in buf-
◦
stored at −40 C until use. Cell debris was removed by
◦
centrifugation at 100,000 × g for 90 min at 4 C. The
supernatant solutions were passed through 0.22 µm-
pore-size filters and were used as a crude cell extract
for enzyme purification or enzyme assays.
2
4
fer with 10% (v/v) glycerol). The most active fractions
eluting at 80 mM KH2PO4) containing salicylate
(
hydroxylase and 1-hydroxy-2-naphthoate hydroxylase
activity were pooled and chromatographed on a Phar-
macia Mono Q HR 5/5 column (bed volume 1 ml, 5 ×
Enzyme assay
◦
All enzymes assays were performed at 30 C us-
5
0 mm) with a linear KH2PO4 (pH 7.5) gradient (20
ing a UVIKON-spectrophotometer 810P (Kontron In-
struments, Germany). One unit of enzyme activity
was defined as the amount of enzyme that con-
verts 1 µmol of substrate per min. Catechol 1,2-
and 2,3-dioxygenase, 2-carboxybenzaldehyde dehyd-
rogenase, 1-hydroxy-2-naphthoate dioxygenase, and
protocatechuate 3,4- and 4,5-dioxygenase activities
were determined as described previously (Hegeman
mM to 200 mM phosphate buffer with 10% (v/v) gly-
cerol). The fractions with salicylate hydroxylase and
1
-hydroxy-2-naphthoate hydroxylase activities (activ-
ity peak at 60 mM KH2PO4) were pooled and were
subjected to chromatography on a Pharmacia Super-
dex 75 Prep-Grade gel filtration column XK16/60 (bed
volume 120 ml, 1,6 × 60 cm). Proteins were eluted
with 50 mM KH2PO4, pH 7.5.
The molecular mass of the protein was measured
by gel filtration chromatography on a Pharmacia Su-
perdex 75 Prep-Grade gel filtration column XK16/60
1
966; Feist & Hegeman 1969; Kiyohara et al. 1976;
Fujisawa & Hayaishi 1968; Ono et al. 1970).
Salicylate and 1-hydroxy-2-naphthoate hy-
droxylase activities were measured at 340 nm by
a modification of the salicylate hydroxylase assay
described by Yamamoto et al. (Yamamoto et al.
(
bed volume 120 ml, 1.6 × 60 cm). The molecu-
lar masses of the standards used for gel filtration
chromatography were 160 kDa (aldolase), 45 kDa
1
965). The reaction mixture (1 ml) contained 100
µM NADH, 100 µl cell extract, 20 mM KH2PO4 (pH
.5) and 50 µM salicylate or 1-hydroxy-2-naphthoate,
respectively. The extinction coefficient used to de-
(
ovalbumin) and 17.8 kDa (myoglobin) (Serva).
7
SDS-polyacrylamide gel electrophoresis
−1
termine the reaction of salicylate was 6.22 mM
The purity of protein preparations was evaluated and
molecular mass of the subunits was determined by so-
dium dodecyl sulfate (SDS)-polyacrylamide gel elec-
trophoresis. It was performed by a modified Laemmli
−1
cm
.
NADH and 1-hydroxy-2-naphthoate both show
significant absorbance at 340 nm. The molar extinc-

1
82
(
Laemmli 1970) procedure on 0.5-mm-thick, 7 cm-
(Vertion 1.05, Elsevier-Biosoft, 1987, Cambridge,
UK).
long vertical slab gels with 12% acrylamide in the
separation gel. The gels were stained with a Bio Rad
silver-staining kit (Pharmacia) by a modification of the
method of Merril et al. (Merril et al. 1981).
Results
Phenanthrene and naphthalene degradation by
Pseudomonas putida BS202-P1
Identification of products
The reaction products were analysed by using an
HPLC system which consisted of a L-4200 UV-Vis
detector, L-6000 pump, a D-2000 chromato-integrator
The wild-type of P. putida BS202 was isolated from
oil-contaminated soil samples after enrichment with
naphthalene and could grow on naphthalene and sali-
cylate but was unable to grow on phenanthrene and
(
Merck Hitachi) and a Grom SIL 100-Octyl-4 FE5mm
reverse phase (4.6 mm × 12.5 cm) column (Grom,
Herrenberg, Germany). Reaction mixtures (1 ml) con-
tained 20 mM KH2PO4 (pH 7.5), 100 µM NADH, and
1
1
-hydroxy-2-naphthoate (Borisoglebskaya & Boronin
983). However, after incubation of this strain for a
period of one month in liquid culture with phenan-
threne, the mutant strain BS202-P1 was isolated. It
simultaneously acquired the ability to grow on phen-
anthrene and 1-hydroxy-2-naphthoate as sole source
of carbon and energy (Balashova et al. 1999).
To test which metabolic pathway the mutant strain
utilized to degrade phenanthrene, several key enzymes
for the degradation of aromatic compounds were de-
termined in cell extracts after growth of the strain
on different carbon sources. Thus, enzyme activities
for the oxidation of catechol and for the oxidation
of NADH in the presence of salicylate or 1-hydroxy-
5
0 µM salicylate or 50 µM 1-hydroxy-2-naphthoate,
respectively. The reactions were started by the addi-
tion of 100 µg of purified enzyme. Reactions were
stopped by acidification with 100 mM H3PO4. Pre-
cipitated protein was removed by centrifugation. The
solvent system for the analysis of salicylate (reten-
tion volume 14.6 ml) and catechol (retention volume
2
0
1
1
.7 ml) consisted of 85% water, 15% methanol and
.2% H3PO4. The solvent system for the analysis of
-hydroxy-2-naphthoate (retention volume 11.1 ml),
,2-dihydroxynaphthalene (retention volume 13.8 ml)
and 1,2-naphthoquinone (retention volume 15.1 ml)
consisted of 80% water, 20% methanol and 0.2%
2
-naphthoate were detected in the cultures grown
on phenanthrene or naphthalene (Table 1). In con-
trast, no o-carboxybenzaldehyde dehydrogenase, 1-
hydroxy-2-naphthoate dioxygenase, protocatechuate
−1
H3PO4. The flow rate was 1 ml min and the absorb-
ance was monitored at 210 nm.
3
,4- and 4,5-dioxygenase activities were found in
Sequencing
these samples (Table 1). This suggested that phenan-
threne was degraded via 1-hydroxy-2-naphthoate and
1
The aminoterminal amino acid sequence was determ-
ined by automated Edman degradation using a model
,2-dihydroxynaphthalene and further by the enzymes
of the naphthalene degradative pathway.
4
70A gas-phase sequencer (Applied Biosystems) in
accordance with the manufacturer’s instructions. The
BLAST network services at the National Center for
Biotechnology Information (Bethesda, Md.) was used
to search protein databases for similar sequences.
Induction experiments
The similarity of the salicylate 1-monooxygenase re-
action and the reaction proposed for 1-hydroxy-2-
naphthoate conversion (Figure 2) and the joint in-
duction of both activities during growth on naph-
thalene and phenanthrene suggested that both reac-
tions could be catalysed by the same enzyme. There-
fore, the induction of salicylate hydroxylase and
1-hydroxy-2-naphthoate hydroxylase activities was
compared after growth on 1-hydroxy-2-naphthoate,
salicylate or after the addition of salicylate (0.1 g
Determination of kinetic parameters
The kinetic values were either determined at a constant
NADH concentration (100 µM) and varying concen-
trations of salicylate or 1-hydroxy-2-naphthoate (0.1
µM − 1 mM) or with a constant concentration of sali-
cylate or 1-hydroxy-2-naphthoate (100 µM) and dif-
ferent NADH concentrations (10 µM to 150 µM). The
Km, Vmax and Ki values were calculated by nonlin-
ear regression analysis using the Enzfitter programm
−1
l
) to succinate-grown cells. Both salicylate and 1-
hydroxy-2-naphthoate hydroxylase activities were de-
tected in crude extract of salicylate and 1-hydroxy-2-

1
83
Table 1. Enzyme activities found in cell extracts of P. putida BS202-P1 after growth on different carbon sources. SH –
salicylate hydroxylase, HH – 1-hydroxy-2-naphthoate hydroxylase, C12O – catechol 1,2-dioxygenase, C23O – catechol
2,3-dioxygenase, 2CBAD – 2-carboxybenzaldehyde dehydrogenase, HO – 1-hydroxy-2-naphthoate dioxygenase, P34O –
protocatechuate 3,4-dioxygenase, P45O – protocatechuate 4,5-dioxygenase
− −1
1
Growth substrate
Specific activity (µmol min mg of protein)
SH
HH
C12O
C23O
2CBAD
HO
P45O
P34O
1
phenanthrene
naphthalene
0.14
0.12
0.11
0.2
–
0.03
0.04
0.05
0.07
–
0.04
0.02
0.01
0.01
–
0.1
–
–
–
–
–
–
–
–
–
1-hydroxy-2-naphthoate
0.06
0.04
0.04
ND
ND
–
–
–
–
salicylate
–
–
–
–
succinate
–
–
–
–
−1
2
succinate + salicylate (0.1 g l
)
0.1
–
0.04
–
ND
ND
ND
ND
ND
ND
ND
ND
ND
−1
succinate + 1-hydroxy-2-naphthoate (0.1 g l
)
ND
1
2
“
–” – activity smaller than 0.005.
ND – not determined.
naphthoate grown and salicylate induced cells. Thus,
salicylate served as a common inductor of salicyl-
ate and 1-hydroxy-2-naphthoatehydroxylase activities
for salicylate and 1-hydroxy-2-naphthoate was detec-
ted when NADPH replaced NADH as cofactor.
Purification of the enzyme
(
2
Table 1). No activities for the salicylate or 1-hydroxy-
-naphthoate dependent oxidation of NADH were
The results shown above suggested that salicyl-
ate 1-monooxygenase might be responsible for
the conversion of 1-hydroxy-2-naphthoate to 1,2-
dihydroxynaphthalene in strain BS202-P1 grown on
phenanthrene. To prove this hypothesis, the salicyl-
ate 1-monooxygenase was further characterized and
purified.
detected in crude extracts of succinate grown cells.
Cofactor requirements
Many monooxygenases contain flavins in their cata-
lytic center (Yamamoto et al. 1965; Beadle & Smith
1
982). It was therefore tested, if the salicylate 1-
Initial studies of salicylate and 1-hydroxy-2-
naphthoate hydroxylase activities with crude extracts
from cells grown on phenanthrene, salicylate or
1-hydroxy-2-naphthoate showed that both activities
were always present with similar relative activities.
Because growth on phenanthrene was very slow and
only low amounts of biomass could be produced,
a crude extract from 1-hydroxy-2-naphthoate grown
cells was used for enzyme purification.
Crude extracts from 1-hydroxy-2-naphthoate
grown cells were fractionated by anion-exchange
chromatography and enzyme activities with 1-
hydroxy-2-naphthoate and salicylate were tested in
the presence of NADH. It was shown that the enzyme
activities for both substrates were found in the same
fractions. Therefore, the enzyme was purified further.
The complete purification confirmed the presence of a
single enzyme responsible for the conversion of both
salicylate and 1-hydroxy-2-naphthoate.
monooxygenase from strain BS202-P1 also required
flavins for activity. Thus, 1 ml of crude extract (4 mg
of protein) was dialyzed at 4 C against 5 l of 20 mM
◦
KH2PO4 buffer (pH 7.5). The preparation lost about
half of its salicylate and 1-hydroxy-2-naphthoate hy-
droxylase activities after five days of dialysis. Then,
FAD, FMN or riboflavin (2 µM each) were added to
aliquots of the dialysed cell extracts and the salicyl-
ate and 1-hydroxy-2-naphthoatehydroxylase activities
were tested after one hour of incubation with the
flavins. The addition of FAD resulted in a complete re-
covery of both enzyme activities. In contrast, no effect
was found after the addition of FMN or riboflavin. The
effects of different FAD concentrations (0.5–4 µM)
were compared and a maximal recovery of the enzyme
activity was found after the addition of 1.5 µM FAD.
It was tested if NADH could be replaced by
NADPH as cofactor for the conversion of salicyl-
ate and 1-hydroxy-2-naphthoate. In the concentration
range tested (50–100 µM), NADH was the preferred
donor of reduction equivalents in respect to both sali-
cylate and 1-hydroxy-2-naphthoate. Only 50% activity
A typical purification procedure is shown in Table
2. The enzyme was purified approximately 25-fold
in three chromatographic steps. Since the enzyme
gradually lost FAD during the purification, it was

1
84
Figure 2. Conversion of salicylate (A) and 1-hydroxy-2-naphthoate (B) by salicylate hydroxylase from P. putida BS202P-1.
routinely reactivated by addition of FAD (1.5 µM)
after every purification step. This resulted in a prepara-
tion that gave a single band upon SDS-polyacrylamide
gel electrophoresis. The molecular mass of the holo-
enzyme was determined by SDS-polyacrylamide gel
electrophoresis and gel-filtration was 45 kDa. Thus
the enzyme appears to be catalytically active as a
monomer.
mixture. NADH concentrations over 100 µM were in-
hibitory (10–150 µM concentrations were tested). A
significant substrate inhibition was observed with 1-
hydroxy-2-naphthoate concentrations >100 µM and
almost no reaction was observed with 1-hydroxy-2-
naphthoate concentrations >500 µM. In contrast, al-
most no substrate inhibition was found with the same
concentrations of salicylate as a substrate (Figure 3).
The kinetic data are presented in Table 3.
Catalytic properties
A number of other compounds were tested as
substrates for the enzyme. A substrate-dependent ox-
idation of NADH was observed with many substituted
salicylates, but 1-hydroxy-2-naphthoate was the only
naphthoic acid, which was converted by the enzyme
The pH optimum of the purified salicylate and
1
-hydroxy-2-naphthoate hydroxylase was 7.0–7.5.
Activities at pH 6.0 and 8.0 were 65 and 96%, respect-
ively, of the maximal activity.
(
Table 4).
The temperature optimum for the hydroxylase
◦
activity was 30 C. The enzyme activities when as-
◦
Products of hydroxylation
sayed at 55, 50, 45, 35, 20 C were 0, 70, 83, 98 and
◦
6
2%, respectively, of that observed at 30 C.
The conversion of salicylate and NADH to catechol
and NAD by the purified enzyme could be analysed by
overlay-spectra, because NADH, catechol and salicyl-
ate were distinguishable by their absorbance maxima
at 340 nm, 260 nm and 296 nm, respectively.
The purified enzyme could be stored in 20 mM
◦
KH2PO4 buffer, pH 7.5 at −20 C without loosing any
activity.
Substrate specificity and inhibition
However, the spectrophotometric analysis of the
conversion of NADH and 1-hydroxy-2-naphthoate
was impeded by the very similar absorption max-
ima of NADH and 1-hydroxy-2-naphthoate. Further-
more, the expected product of the reaction, 1,2-
dihydroxynaphthalene, and its autoxidation product,
To compare the catalytic efficiencies of the enzyme
with both substrates, different concentrations of sali-
cylate and 1-hydroxy-2-naphthoate were converted by
the purified enzyme. 100 µM NADH concentration
was used to provide excess of cofactor in reaction

1
85
−1
−1
Figure 3. Michaelis-Menten plots of salicylate hydroxylase (A) and 1-hydroxy-2-naphtoate (B) activities. V is presented in µmol min mg .
Table 2. Copurification of salicylate and 1-hydroxy-2-naphthoate hydroxylase activities from P. putida BS202-P1. SH – salicylate
hydroxylase, HH – 1-hydroxy-2-naphthoate hydroxylase
Purification step
Protein
mg)
Vol.
(ml)
Total activity
SH HH
Specific activity
(U/mg)
Recovered activity
(%)
Purification factor
(
SH
HH
SH
HH
SH
1
HH
Cell extract
Resource Q
MonoQ
22.8
3.6
6
4
2
4
5.3
2.8
2.4
0.4
1.6
0.8
0.2
0.7
12
0.07
0.17
4.5
100
52
100
51
1
2.4
64
21
3.5
0.2
0.9
45
58
60
25
Superdex75
0.08
0.12
5
1.5
7.5
7.7

1
86
Figure 4. Alignment of the N-terminal amino acid sequence of salicylate and 1-hydroxy-2-naphthoate hydroxylase from P. putida BS202-P1
with sequences for salicylate 1-monooxygenases from P. stutzeri AN10 (NahG AN10), P. putida KF715, P. putida PpG7, P. putida NCIB9816
(NCBI accession number X83926, deduced from nucleotide sequence data), Sphingomonas sp. AJ1 (NCBI accession number AB000564,
deduced from nucleotide sequence data). Positions identical with those in the P. putida BS202-P1 are shown on a grey background.
Table 3. Kinetic data of the purified salicylate 1-monooxygenase
Substrate
Salicylate
K
Vmax
−1
µM) (µmol min mg
Vmax/KM
K
i
M
−1
(
(µM)
1.72
4.01
0.27
0.12
0.16
0.03
935
127
1-Hydroxy-2-naphthoate
1
,2-naphthoquinone, show some absorbance at 340
Pseudomonas putida NCIB9816 (13 aa from 22 were
nm (Kuhm et al. 1991). Therefore, the products
from the conversion of salicylate and 1-hydroxy-
identical).
2
-naphthoate with NADH were identified by using
HPLC. This assay indicated that the purified en-
zyme converted salicylate to a substance with a
retention volume corresponding to catechol and 1-
hydroxy-2-naphthalene to a substance with a retention
volume corresponding to 1,2-dihydroxynaphthalene.
Discussion
The enzymes of the upper part of the naphthalene
pathway have been shown earlier to possess a broad
substrate specificity. Thus the enzymes from Pseudo-
monas sp. NCIB 9816 were able to convert an-
thracene to 2-hydroxy-3-naphthoate and phenanthrene
to 1-hydroxy-2-naphthoate (Menn et al. 1993; Kiy-
ohara et al. 1994). It has been repeatedly pro-
posed that 1-hydroxy-2-naphthoate could be con-
verted to 1,2-dihydroxynaphthalene and then fur-
ther metabolized by the same enzymes which also
convert 3,4-dihydroxyphenanthrene to 1-hydroxy-2-
naphthoate. Nevertheless, before our study there was
no information available which enzyme was respons-
ible for the conversion of 1-hydroxy-2-naphthoate
to 1,2-dihydroxynaphthalene in those phenanthrene-
degrading strains which metabolize phenanthrene via
1,2-dihydroxynaphthalene. This might be one of the
limiting steps which prevent growth of many naph-
thalene degrading bacteria on phenanthrene (Kiyohara
et al. 1994, Balashova et al. 1999).
1
,2-Naphthoquinone was also detected in the reac-
tion mixture. This is presumably due to the spon-
taneous autoxidation of 1,2-dihydroxynaphthalene to
1
,2-naphthoquinone.
N-terminal sequence
The amino-terminal amino acid sequence of the sali-
cylate and 1-hydroxy-2-naphthoate converting en-
zyme was determined and compared with those of
other salicylate hydroxylases. This analysis indic-
ated that the enzyme from BS202-P1 differed from
a previously studied salicylate 1-monooxygenase, but
was clearly related to other salicylate hydroxylases
(
Figure 4). The amino acid sequence of the purified
protein showed the highest degree of sequence con-
servation with the salicylate-1-monooxygenase from

1
87
Table 4. Substrate specificity of the purified salicyl-
ate 1-monooxygenase
The determination of the aminoterminal amino
acid sequence and the comparison with previously
determined sequences demonstrated that the salicyl-
ate 1-monooxygenase from strain BS202-P1 showed
high degree of homology to other salicylate 1-
monooxygenaes (59–49%). The holoenzyme from P.
putida BS202-P1 showed a similar size (45 kDa) as
the salicylate 1-monooxygenases from P. putida S-
Substrate
Salicylate
Relative activity (%)
100
30
63
39
40
110
110
83
67
<5
85
114
78
<5
<5
30
3
4
5
3
4
5
5
3
5
2
2
2
1
2
1
2
2
-Chlorosalicylate
-Chlorosalicylate
-Chlorosalicylate
-Aminosalicylate
-Aminosalicylate
-Aminosalicylate
-Methylsalicylate
-Methylsalicylate
-Sulfosalicylate
,3-Dihydroxybenzoate
,5-Dihydroxybenzoate
,6-Dihydroxybenzoate
-Naphthoate
1
and P. putida PpG7 (Yamamoto et al. 1965; You
et al. 1990), but differed from the dimeric salicylate
1-monooxygenase described for Burkholderia cepa-
cia ATCC 29351 and ATCC 29352 (White-Stevens &
Kamin 1972; Wang et al. 1984).
The purified salicylate 1-monooxygenase conver-
ted a broad spectrum of substituted salicylates. This
was also previously observed for other salicylate hy-
droxylases from P. putida S-1, Pseudomonas stutzeri
AN10 and Burkholderia cepacia ATCC 29352 strains
-Naphthoate
-Hydroxy-2-naphthoate
-Hydroxy-3-naphthoate
-Hydroxy-1-naphthoate
<5
<5
(
Yamamoto et al. 1965; Bosch et al. 1999; White-
The enzyme activity was determined in a reaction
mixture (1 ml) which contained 20mM KH PO
Stevens & Kamin 1972). The purified enzyme oxid-
ized from naphthoic acids tested only 1-hydroxy-2-
naphthoate with significant rates (Table 4). This might
be the reason why the strain BS202-P1 could not util-
ize other PAHs, in particular anthracene, which is
metabolized via 2-hydroxy-3-naphthoate.
2
4
(pH 7.5), 100 µM NADH, 50 µM of the respective
substrate and 20 µg of purified enzyme. The ox-
idation of NADH was determined at 340 nm. The
enzyme activity with salicylate (0.9 U/mg) was taken
as 100%.
The results of the present study suggested that
salicylate 1-monooxygenases are responsible for the
oxidation of 1-hydroxy-2-naphthoatein phenanthrene-
degrading fluorescent pseudomonads. The attempts to
adapt the naphthalene-degrading fluorescent pseudo-
monads from our collection to growth on phenan-
threne showed that most of these strains could be
adapted to grow on phenanthrene after a certain period
of incubation. In addition to the ability to grow
on phenanthrene these bacteria became capable of
growing on 1-hydroxy-2-naphthoate. Nevertheless, it
was shown that the relative activities of cell extracts
from the naphthalene degrading isolates and their
phenanthrene-degrading derivatives for the conversion
of salicylate and 1-hydroxy-2-naphthoate were almost
identical (data not presented).
This suggested that the induction of the up-
per and lower naphthalene degradative pathway may
limit the degradation of phenanthrene by fluorescent
pseudomonads. For Pseudomonas sp. NCIB 9816
and Pseudomonas putida G7 it was previously shown
that salicylate (substituted salicylates) could serve as
inductors for the relevant enzymes (Barnsley 1975;
Shamsuzzaman & Barnsley 1974a; Shamsuzzaman &
Barnsley 1974b; Schel 1985). This indicates that dur-
ing the slow turn-over of phenanthrene by naphthalene
The results of the present manuscript demon-
strate that after growth on 1-hydroxy-2-naphthoate
or salicylate strain P. putida BS202-P1 synthesized
a single salicylate 1-monooxygenase, which conver-
ted 1-hydroxy-2-naphthoate under the standard re-
action conditions with 30% of the activity found
with salicylate. The previously studied salicylate 1-
monooxygenases from two different bacteria which
had been enriched with salicylate also showed some
NADH-oxidation after the addition of 1-hydroxy-2-
naphthoate but with considerably lower relative rates
compared to salicylate (vrel salicylate: 1-hydroxy-
2
(
1
-naphthoate 100: 1.9 and 100: 15, respectively)
Yamamoto et al. 1965; White-Stevens & Kamin
972). Salicylate 1-monooxygenases are known to
perform with certain substrate-analogues (so called
pseudo-substrates) NADH-oxidation reactions which
do not result in the hydroxylation of the substrates
(
White-Stevens & Kamin 1972). Thus, the detection
of the formation of 1,2-dihydroxynaphthalene from
-hydroxy-2-naphthoate by the purified salicylate 1-
1
monooxygenase gave the first conclusive evidence
that this enzyme could indeed catalyse the productive
metabolism of 1-hydroxy-2-naphthoate and salicylate.

1
88
degrading fluorescent pseudomonads only very low
amounts of salicylate are accumulating which do not
result in a sufficient induction of the enzymes involved
in the degradation of naphthalene and phenanthrene.
To elucidate what genetic alterations are necessary
to acquire the ability to grow on phenanthrene in
naphthalene-degrading strains will be the subject of
further investigations.
Evans CGT, Herbert D & Tempest DB (1970) The continuous
cultivation of microorganisms. 2. Construction of a chemostat.
Meth. Microbiol. 2: 277–327
Hegeman GD (1966) Synthesis of the enzymes of the mandelate
pathway by Pseudomonas putida. I. Synthesis of enzymes of the
wild type. J. Bacteriol. 91: 1140–1154
Iwabuchi T & Harayama S (1998) Biochemical and molecular
characterization of 1-hydroxy-2-naphthoate dioxygenase from
Nocardioides sp. KP7. J. Biol. Chem. 273: 8332–8336
Kiyohara H, Nagao K & Nomi R (1976) Degradation of phenan-
threne through o-phthalate by Aeromonas sp. Agric. Biol. Chem.
40: 1075–1082
Kiyohara H & Nagao K (1978) The catabolism of phenanthrene and
naphthalene by bacteria. J. Gen. Microbiol. 105: 69–75
Kiyohara H, Torigoe S, Kaida N, Asaki T, Iida T, Hayashi H & Tak-
izawa N (1994) Cloning and characterisation of chromosomal
gene cluster, pah, that encodes the upper pathway for phen-
anthrene and naphthalene utilisation by Pseudomonas putida
OUS82. Biochem. J. 176: 2439–2443
Kuhm AE, Stolz A, Ngai K-L & Knackmuss H-J (1991) Purification
and characterization of a 1,2-dihydroxynaphthalene dioxygenase
from a bacterium that degrades naphthalenesulfonic acids. J.
Bacteriol. 173: 3795–3802
Acknowledgments
This study was supported by ESF GPoll/CRP/9811
grant, INTAS Fellowship grant for Young Scientists
YSF 98-97 and partly supported by a grant from
the Russian Federal Research and Technical Pro-
gram “Novel Methods of Bioengineering” within the
trend “Environmental Biotechnology”. The authors
thank Dr. Andrea Kuhm, Universität Stuttgart (Ger-
many), for help in enzyme purification, Dr. Volker
Noedinger, Universität Stuttgart (Germany), for carry-
ing out N-terminal aminoacid analysis of the purified
hydroxylase and Anatoly Koshelev, Institute of Bio-
chemistry and Physiology of Microorganisms (Rus-
sia), for biomass preparation.
Laemmli UK (1970) Cleavage of structural proteins during the as-
sembly of the head of bacteriophage T4. Nature (London) 227:
680-685
Lee J, Oh J, Min KR & Kim Y (1996). Nucleotide sequence of
ꢀ
salicylate hydroxylase gene and its 5 -flanking region of Pseudo-
monas putida KF715. Biochem. Biophys. Res. Comm. 218:
544–548
Menn F-M, Applegate BM & Sayler GS (1993) NAH plasmid-
mediated catabolism of anthracene and phenanthrene to naph-
thoic acids. Appl. Environ. Microbiol. 59: 1938–1942
Merril CR, Goldman D, Sedman SA & Ebert MH (1981) Ul-
trasensitive stain for proteins in polyacrylamide gels shows in
cerebrospinal fluid proteins. Science 211: 1437–1438
Ono K, Nosaki M & Hayaishi O (1970) Purification and some prop-
erties of protocatechuate 4,5-dioxygenase. Biochim. Biophys.
Acta 220: 224–238
Schell MA (1985) Transcriptional control of the nah and sal
hydrocarbon-degradation operons by the nahR gene product.
Gene 36: 301–309
Shamsuzzaman KM & Barnsley EA (1974a) The regulation of
naphthalene oxygenase in pseudomonads. J. Gen. Microbiol. 83:
References
Balashova NV, Kosheleva IA, Golovchenko NP & Boronin AM
(1999) Phenanthrene metabolism by Pseudomonas and Burkhol-
deria strains. Process Biochem. 35: 291–296
Barnsley EA (1975) The induction of the enzymes of naph-
thalene metabolism in pseudomonads by salicylate and 2-
aminobenzoate. J. Gen. Microbiol. 88: 193–196
Beadle TA & Smith ARV (1982) The purification and properties of
2,4-dichlorophenol hydroxylase from a strain of Acinetobacter
species. Eur. J. Biochem. 123: 323–332
165–170
Borisoglebskaya AN & Boronin AM (1983) Population changes of
the Pseudomonas putida BS202 strain containing plasmid NPL-1
on capability for naphthalene catabolism. Russ. J. Microbiol. 53:
Shamsuzzaman KM & Barnsley EA (1974b) The regulation of
naphthalene metabolism in pseudomonads. Biochem. Biophys.
Res. Comm. 60: 582–589
Stringfellow TW & Aitken DM (1994) Comparative physiology of
phenantrene degradation by two dissimilar pseudomonads isol-
ated from a creosote-contaminated soil. Can. J. Microbiol. 40:
301–306
Bosch R, Moore ERB, Garcia-Valdes E & Pieper DH (1999) NahW,
a novel, inducible salicylate hydroxylase involved in mineraliza-
tion of naphthalene by Pseudomonas stutzeri AN10. J. Bacteriol.
432–438
181: 2315–2322
Wang L-H, Tu S-C & Lusk RC (1984) Apoenzyme of Pseudomonas
cepacia salicylate hydroxylase. J. Biol. Chem. 259: 1136–1142
White-Stevens RH & Kamin H (1972) Studies of a flavoprotein,
salicylate hydroxylase. I. Preparation, properties, and the un-
coupling of oxygen reduction from hydroxylation. J. Biol. Chem.
Bradford MM (1976) A rapid and sensitive method for the quantita-
tion of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72: 248–254
Feist CF & Hegemann GD (1969) Phenol and benzoate metabolism
by Pseudomonas putida: regulation of tangential pathways. J.
Bacteriol. 100: 869–877
247: 2358–2370
Yamamoto S, Katagiri M, Maero H & Hayaishi O (1965) Sali-
cylate hydroxylase, a monooxygenase requiring flavin adenin
dinucleotide. J. Biol. Chem. 240: 3408–3413
You I-S, Murray RI, Jollie D & Gunsalus IC (1990) Purification and
characterization of salicylate hydroxylase from Pseudomonas
putida PpG7. Biochem. Biophys. Res. Comm. 169: 1049–1054
Fujisawa H & Hayaishi O (1968) Protocatechuate 3,4-dioxygenase.
I. Crystallization and characterization. J. Biol. Chem. 243: 2673–
2681
Evans WC, Fernley HN & Griffiths E (1965) Oxidative metabolism
of phenanthrene and anthracene by soil pseudomonads: the ring-
fission mechanism. Biochem. J. 95: 819–831