Phenol hydroxylase from
Acinetobacter radioresistens
S13
Isolation and characterization of the regulatory component
Ersilia Griva
1
, Enrica Pessione
1
, Sara Divari
1
, Francesca Valetti
1
, Maria Cavaletto
2
, Gian Luigi Rossi
3
and Carlo Giunta
1
1
Dipartimento di Biologia Animale e dell’Uomo, Universita
`
di Torino, Italy;
2
Dipartimento di Scienze e Tecnologie Avanzate,
Universita
`
del Piemonte Orientale, Alessandria, Italy;
3
Dipartimento di Biochimica e Biologia Molecolare,
Universita
`

Keywords: regulatory proteins; multicomponent mono-
oxygenase; phenol hydroxylase.
Acinetobacter radioresistens S13 is able to grow on phenol
as the sole carbon and energy source via the ortho-pathway
(b-ketoadipate pathway). The first enzyme involved in
phenol degradation is phenol hydroxylase (PH), a mono-
oxygenase utilizing NADH as electron donor.
In previous studies we have found that the enzyme is
composed of three moieties which are readily separated by
chromatographic steps: the oxygenase (PHO), composed of
two heterotrimers (abc) (S. Divari, F. Valetti, P. Caposio, E.
Pessione, M. Calvaletto, E. Griva, G. Gribaudo, G. Gilardi
& C. Giunta, unpublished observation), the reductase
(PHR) [1] and a third protein (PHI) that is described in
this work.
A similar molecular composition has been found in
phenol hydroxylases from Pseudomonas CF 600 [2] and
A. calcoaceticus NCIB 8250 [3] and in toluene-2-mono-
oxygenase from Burkholderia cepacia [4], as well as in the
soluble methane monooxygenases (MMOs) from Methylo-
coccus capsulatus [5], Methylosinus trichosporium [6], Meth-
ylocystis sp.M [7] and in alkene monooxygenase from
Nocardia corallina [8].
In phenol hydroxylase of A. radioresistens S13, the third
component is needed for the overall enzyme activity; in
phenol hydroxylase from Pseudomonas CF 600, it promotes
substrate–oxygenase interaction [9]; in MMOs it alters the
local environment and the redox potential of the catalytic
centre [6,10–12].
Interestingly, in other aromatic monooxygenases (i.e.

ting substrate-binding to the active site of PHO; (b) PHI–
PHR interaction, possibly resulting in an altered confor-
mation of PHR more suitable for electron transfer to
PHO; (c) PHI–PHO interaction, possibly causing a
conformational change leading to the opening of the
PHO active site.
Materials and methods
Bacterial strain
The A. radioresistens S13 strain used in this work was
isolated as previously described [16,17]. This bacterium
bears several natural plasmids and is able to grow on either
phenol or benzoate as the only carbon source.
Culture conditions
The culture media used were Luria-Bertani (LB) broth
(peptone 10 gÆL
)1
,NaCl10 gÆL
)1
, yeast extract 5 gÆL
)1
)and
the Sokol and Howell [18] minimal medium, where phenol
was the only carbon source. The fed-batch fermentation
procedure was used. The acclimation method was the same
as previously reported [19]. Cells were harvested when
growth reached the stationary phase and were stored frozen
()80 °C).
Preparation of crude extract
Cells were washed twice in 50 m
M

Both in the crude extract and after separation from the
oxygenase, PHR activity was monitored by the reduction of
cytochrome c in the presence of NADH at 550 nm [1].
Protein determination
Protein content was determined by the Bradford test [20],
using bovine serum albumin as standard.
PHI purification
An anion exchange DE-52 cellulose column (Whatman)
(2.6 · 20 cm) was equilibrated with 50 m
M
Hepes/NaOH
buffer, pH 7.0. The crude extract was eluted with a 0–0.5
M
sodium sulfate gradient in 50 m
M
Hepes/NaOH buffer,
pH 7.0 (final volume 1.1 L). This procedure allowed us to
separate the oxygenase moiety. Fractions showing reduc-
tase activity were applied on a second anion exchange
column Source Q15 (Pharmacia) (1 · 5 cm) equilibrated
with 50 m
M
Hepes/NaOH buffer, pH 7.0 containing
0.05
M
sodium-sulfate. PHR and PHI were coeluted from
this column with a 0.05–0.5
M
sodium sulfate gradient in
50 m

Hydrophobic interaction chromatography
In order to inquire whether PHI could interact directly with
phenol, PHI was dissolved in 50 m
M
Hepes/NaOH buffer,
pH 7.0, containing 0.15
M
sodium sulfate and was loaded
on a Phenyl-Sepharose column (2.5 · 8cm)(Pharmacia)
equilibrated in the same buffer. The flow rate was
2mLÆmin
)1
.
Molecular mass determination
The molecular mass was determined by means of SDS/
PAGE, size exclusion chromatography and mass spectro-
metry.
SDS/PAGE was carried out in separating gels containing
15% acrylamide. The following proteins were used as
standards: phosphorylase B (97 kDa), bovine serum albu-
min (67 kDa), ovalbumin (45 kDa), carbonic anhydrase
(31 kDa), trypsin inhibitor (21 kDa) and lysozyme
(14 kDa). In addition, molecular mass peptide standards
(Pharmacia) were used: globin (16.9 kDa), globin I + II
(14.4 kDa), globin I + III (10.7 kDa) and globin I
(8.2 kDa). Proteins were detected by silver staining.
A Superdex 75-FPLC column (2.6 · 60 cm) (Pharma-
cia) was equilibrated with 50 m
M
Hepes/NaOH buffer,

represent the elution, void and total column
volume, respectively. The same experiment was repeated
using 50 m
M
Hepes/NaOH buffer, pH 7.0, as eluent.
PHI molecular mass was confirmed by matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF)
mass spectral analysis, using a Biflex mass spectrometer
(Bruker). The sample (3 nmol) was desalted, lyophilyzed and
resuspended in 50 lL acetonitrile/water solution (70 : 30,
v/v) and mixed with 50 lL sinapinic acid matrix. One lL
of the resulting solution ( 30 pmol of PHI) was loaded.
Isoelectric focusing
The isoelectric point was determined by analytical IEF
electrophoresis (Phast System, Pharmacia); the markers
were those supplied by Pharmacia (pI calibration kit).
NH
2
-terminal sequence
After SDS/PAGE, the protein band was blotted onto
Immobilon P (Millipore) membrane. The N-terminus was
sequenced using the Applied Biosystems 470A automatic
microsequencer, following the Edman degradation [22].
Optical spectroscopy
The UV/Vis absorption spectrum of purified protein in
50 m
M
Hepes/NaOH buffer, pH 7.0, was determined from
200 to 700 nm using a DU-70 Spectrophotometer (Beck-
man), at 20 °C. Fluorescence emission spectra of protein in

modified from Lovenberg [23] and Beinert [24], respectively.
Kinetic constants
The catalytic activity of PHR was evaluated both in the
presence and in the absence of PHI.
K
m
and k
cat
were determined from Hanes–Haldane plot
for the two electron acceptors cytochrome c and NBT,
using 0.24 m
M
NADH as electron donor in 50 m
M
Tris/
sulfate buffer, pH 8.5, at 30 °C.
Reconstitution of PH activity ‘
in vitro
’
Reconstitution of the complex from the purified fractions
was studied by investigating the overall PH activity in the
presence of variable amounts of each component. The assay
was performed with a Clark type electrode in the presence
of 1.7 m
M
NADH in 100 m
M
Mops/NaOH buffer, pH 7.4
at 24 °C. The basal oxygen consumption was subtracted
from the consumption recorded after addition of 1 m

PH activity.
The yield of the PHI component suggested that it
accounts for 0.25–0.3% of the soluble cellular protein.
Molecular mass and isoelectric point
The molecular mass of PHI, determined by SDS/PAGE,
was 10 kDa. A similar result (8.8 kDa) was obtained by
mass spectrometry (MALDI-TOF) analysis (Fig. 2). A
twice as large value (18 kDa) was found by gel-permeation
chromatography on Superdex 75. Therefore, it is likely that
the native protein occurs as a dimer. The isoelectric point,
determined by analytical isoelectrofocusing on ampholyte
gels, was 4.1.
Absence of redox centres
The UV/Vis absorption spectrum of PHI at pH 7.0 and at
20 °C exhibited the typical protein peak at 280 nm.
Neither in native samples nor in samples treated with
reducing or oxidizing agents were detected chromophoric
groups absorbing in the interval between 300 and 800 nm.
These results were confirmed by the Lovenberg [23] and
Beinert analyses [24] which failed to show iron- or sulfur-
containing redox-centres in the pure protein. In agreement
with these findings, the emission spectrum of PHI,
determined by spectrofluorimetry in the same conditions,
exhibited a maximum at 345 nm on excitation at either 280
or 295 nm.
N-terminal sequence
The first 11 aminoacids at the N-terminus of PHI (sequence:
SKVYLALQDND) were compared with the sequences of
the so called ‘intermediate components’ from two other
PHs. The N-terminal sequence of PHI from A. radioresis-

PHI is essential for the catalytic activity
of the reconstituted PH system
A stoichiometry 2 PHR monomers: 1 PHI dimer: 1 PHO
(abc) dimer was found to provide optimal phenol reaction
rates.
PH activity in function of PHR concentration follows a
Michaelian behaviour at fixed concentrations of PHO and
PHI (Fig. 4). When the latter components are present at
0.6 l
M
, in terms of dimeric units, a maximum turnover
number of 70 min
)1
was obtained upon increasing PHR
concentration: the plateau is reached at 1.2 l
M
PHR (in
terms of monomeric units) (Fig. 4, continuous line with
triangles). Excess of PHI over PHO does not alter the
overall enzyme activity (Fig. 4, broken line with asterisks),
in contrast to what observed in MMO from Methylosinus
trichosporium [26].
The emission intensity of the PHR flavin shows a 17%
increase after addition of PHI to either the complex PHR-
PHO or to PHR alone, in the stoichiometry ratio
PHR : PHO : PHI 2 : 1 : 1 (Fig. 5). On the contrary, the
emission spectrum of PHO-bound ANS is not affected by
the addition of PHI, suggesting no specific PHI interaction
with the substrate binding site of PHO.
PHI is not required for PHR activity towards alternative

Fig. 4. Reconstitution of PH activity in vitro in the presence of variable
amounts of each component. The assay was performed with a Clark-
type electrode in the presence of 1.7 m
M
NADH in 100 m
M
Mops/
NaOHbuffer,pH 7.4,at24 °C. Data were obtained with 1 m
M
phenol
as a substrate and are corrected by subtraction of the basal oxygen
consumption. PHI concentration (0.3, 0.6 and 1.2 l
M
, i.e. PHI/PHO
ratios: 0.5, 1 and 2) was varied over a range of PHR/PHO ratios (up to
6), keeping fixed a PHO concentration of 0.6 l
M
. The data were fitted
to Michaelis–Menten curves. Squares and dotted line: data and fitting
with PHI/PHO ratio of 0.5. Triangles and continuous line: data and
fitting with PHI/PHO ratio of 1. Asterisks and broken line: data and
fitting with PHI/PHO ratio of 2.
Fig. 5. Effect of PHI on the flavin fluorescence of the complex PHR–
PHO. Dotted line: fluorescence emission spectrum of the couple PHR–
PHO (2 : 1) in Hepes/NaOH buffer, pH 7.0. Solid line: fluorescence
emission spectrum after the addition of PHI to the above mentioned
mixture. k excitation 450 nm.
Fig. 3. Temperature dependence of PHI far-UV circular dichroism spectra. Conditions: 10 l
M
PHI in 10 m

emission spectrum of PHI. Moreover, PHI does not bind
ANS (a probe for hydrophobic sites) and is not retained by
the phenyl Sepharose column (a ligand for phenolic-
recognizing sites and for hydrophobic sites in general).
These results differ from those reported for the regulatory
protein P2 of Pseudomonas CF600 phenol hydroxylase [9],
a molecule with an N-terminus sequence very similar to
that of PHI. NMR studies on P2 have suggested the
presence of a hydrophobic cavity [9] that is likely to bind
phenol and thus favour its interaction with the oxygenase
moiety. The data here reported do not provide any
evidence for the presence of a phenol-binding or other
hydrophobic sites. However, we cannot exclude binding of
the aromatic substrate to a buried cavity in case such an
interaction would not cause changes in the protein
fluorescence signal.
An interaction between PHI-PHR is a likely candidate
to explain the regulatory effect. In fact, on addition of PHI,
the fluorescence of PHR-bound flavin increases. This
finding points to a PHI-induced conformational change
of PHR, possibly resulting in a more pronounced exposure
of FAD to the aqueous solvent. The most important
functional consequence of this PHI-induced conformational
transition of PHR might be: (a) a better exposure of the
Fe/S cluster involved in the electron transfer to PHO; (b) a
favourable orientation of a specific PHR domain allowing
for optimal interaction with PHO. If the former hypothesis
were true, one could expect a more efficient electron
transfer not only to PHO but also to artificial electron
acceptors. However, the reduction of either cytochrome c

as shown by the lack of alteration in the PHO-ANS
fluorescence upon addition of PHI.
In summary, while the regulatory components of MMOs
act via an interaction with the oxygenase [28–30], and, in the
case of Pseudomonas CF600 phenol hydroxylase, via a
direct interaction with the substrate itself [9], in the case of
A. radioresistens S13 phenol hydroxylase, PHI appears to
interact with the reductase moiety. This PHI–PHR interac-
tion promotes the PHR conformational changes that are
necessary to optimize the mutual orientation of PHR and
PHO and thus electron transfer between them.
Acknowledgements
This work is supported by the EC Biotechnology programme, contract
BIO-960413. We are grateful to D. Corpillo (University of Turin) for
mass spectroscopy analysis, to A. Conti and G. Giuffrida (CNR-
Torino) for N-terminal sequence determination and to D. Cavazzini
(University of Parma) for helpful discussion and CD technical
assistance.
Table 1. Catalytic parameters of A. radioresistens S13 PHR, alone and in the presence of PHI, determined with two artificial electron acceptors. The
K
m
and k
cat
values were determined at 30 °C, in 50 m
M
Tris/sulfate buffer, pH 8.5, using NADH as the electron donor.
Cytochrome c NBT
K
m
(l

)1
)
PHR 1.3 ± 0.3 61 ± 6 47 10 ± 3 0.63 ± 0.08 0.063
PHR + PHI 1.5 ± 0.2 55 ± 7 36 9 ± 3 0.66 ± 0.05 0.070
Ó FEBS 2003 A. radioresistens S13 phenol hydroxylase regulatory component (Eur. J. Biochem. 270) 1439
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