(Received for publication, November 29, 1995; and in revised form, January 30, 1996)
From the
Biphenyl (BPH) dioxygenase oxidizes BPH to
2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein
(ISP), a ferredoxin FER
, and a ferredoxin
reductase RED
. RED
and FER
transfer electrons from NADH to an Fe-S active center of ISP
which activates molecular oxygen for insertion into the
substrate. In this work B-356 ISP
complex and its
and
subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356
ISP
construction carrying a single His tail on the
N-terminal portion of the
subunit was active. Its major features
were compared to the untagged enzyme. In both cases, the native form is
an
heteromer, with each
unit containing a [2Fe-2S] Rieske center (
= 8,300 M
cm
) and a mononuclear Fe
.
Although purified His-tagged
subunit showed the characteristic
absorption spectra of Rieske-type protein, reassociation of this enzyme
component and His-tagged
subunit to reconstitute active
ISP
was weak. However, when His-tagged
and
subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISP
could be
purified on Ni-nitrilotriacetic acid resin.
Biphenyl dioxygenase (BPH dox) ()catalyzes the first
step of the bacterial BPH degradation pathway. The enzyme introduces
molecular oxygen into the ortho-meta positions on one of the
aryl rings to generate 2,3-dihydro-2,3-dihydroxybiphenyl. In a previous
study(1) , we have reported the purification and
characterization of Comamonas testosteroni strain B-356-BPH
dox system. The enzyme comprises three components which are: the
terminal oxygenase, an iron-sulfur protein (ISP
) made up
of an
-subunit (M
= 51,000) and a
-subunit (M
= 22,000), encoded by bphA and bphE, respectively; a ferredoxin
(FER
, M
= 12,000) encoded by bphF; and a ferredoxin reductase (RED
, M
= 43,000) encoded by bphG.
FER
and RED
were found to be involved in
electron transfer from NADH to ISP
(1) . The
Rieske center of the oxygenase component is then believed to receive
the electron and pass it to a mononuclear Fe
which
activates molecular oxygen for insertion into the
substrate(2, 3) .
The ISP component
has been purified from BPH-induced bacteria of strain B-356 (1) and from Pseudomonas sp. LB400 (4) . Since
active purified FER
and RED
were difficult
to obtain from cell extracts of parental
strains(1, 4) , these enzyme components were purified
from Escherichia coli recombinant clones using the His-bind
QIAGEN system(1) . Both His-tagged FER
and
His-tagged RED
from strain B-356 were able to transfer
electrons from NADH to B-356-ISP
. However, purification
of the individual active ISP
and
subunits has
not yet been reported.
Understanding the various factors that
contribute to the strain selectivity pattern toward substrate should
help the modeling of new mutants with increased ability to degrade BPH
analogs such as polychlorinated biphenyls. The BPH dox reactivity
pattern is a major determinant affecting the performance of bacterial
polychlorinated biphenyl degraders. The BPH dox-congener selectivity
pattern is partly affected by the position of attack on the aromatic
ring. For example, the capacity of Pseudomonas sp. LB400 to
preferentially degrade the ortho-substituted polychlorinated
biphenyl congeners was attributed to its ability to oxygenate BPH at
ring positions 3 and 4 in addition to 2 and 3. Haddock et al. (5) have recently shown that partially purified LB400-BPH dox
was able to attack 2,2`,5,5`-tetrachlorobiphenyl (for which there is no
available ortho-meta sites for oxygenation) in a
3,4-position suggesting that the same enzyme catalyzes both type of
attacks. Using site-directed mutagenesis, Erickson and Mondello (6) have provided evidence that minor structural differences of
the ISP
subunit component are responsible for major
changes in the reactivity pattern of strain LB400-BPH dox.
Although the substrate selectivity of strain B-356 distinguishes it from strain LB400, we have recently shown that strain B-356 dox is also able to oxygenate BPH at both 2,3- and 3,4-positions(1) .
Other
studies have suggested that additional determinants are affecting the
reactivity pattern of various strains toward BPH analogs. For example,
Hirose et al. (7) have found that the BPH dox
components of Pseudomonas pseudoalcaligenes KF707 are to some
extent, interchangeable with those of the Pseudomonas putida F1-toluene dioxygenase system encoded by the tod operon.
Several of the recombinant enzymes tested were found to manifest new
degradative abilities that neither of the original enzymes possessed.
Their results suggest that the oxygenase subunit is specific to
the system to which it belongs. However, to date, the role of the
ISP
subunit in enzyme activity remains unknown.
At this time, it is clear that further investigation is required at
the molecular level to identify the role of the BPH dox components
regarding catalytic activity and substrate selectivity. However,
because most of these enzyme components are unstable and cannot sustain
the large number of manipulations needed for their purification, new
approaches are needed to obtain purified active enzyme. In this work we
have identified some of the major features of purified strain B-356
His-tagged ISP and of individual His-tagged ISP
subunits. We have also found that the His-tagged ISP
subunits can be reassembled in vitro to produce an
active oxygenase component.
Plasmid DNA from E. coli, restriction endonuclease digestions, ligations, agarose gel electrophoresis, and transformation of E. coli cells were done according to protocols described by Sambrook et al.(9) . Polymerase chain reactions (PCR) were performed using Pwo DNA polymerase following the method given by Boehringer Mannheim.
The PCR
products were digested with BamHI and KpnI. A
1.9-kilobase DNA fragment (for bphAE), a 1.3-kilobase DNA
fragment (for bphA), and a 0.6-kilobase DNA fragment (for bphE) containing the entire coding sequences were isolated and
cloned into the compatible sites of pQE31. Constructions were such that
the His tail added 13 amino acids (MRGSHHHHHHTDP) to the protein at its
N-terminal portion. When both the and
subunits were
produced together in the same clone, the His tail was attached to the
subunit only.
The iron content of protein was evaluated by the ferrozine colorimetric method modified by Batie et al.(13) . Acid-labile sulfide was determined according to the method of Fogo and Popowsky (14) as modified by King and Morris(15) . Protein concentrations were estimated by the methods of Lowry (16) and Bradford (17) using bovine serum albumin as standard.
Occasionally BPH dox activity was evaluated spectrophotometrically
at 434 nm in a coupled reaction in the presence of excess amounts of
purified 2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase (B2,3D)
and 2,3-dihydroxybiphenyl 1,2-dioxygenase (B1,2O). All other conditions
were identical to the one described above. The used
for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage product of BPH, was 22,000 M
cm
. The steps used to
obtain the purified B1,2O preparation were presented previously
(Genetics of Industrial Microorganisms, June 26 to July 1, 1994,
Montreal, Quebec, Canada, abstract no. p. 264). The purification scheme
for B2,3D will be described elsewhere.
The yield of purified
His-tagged ISP was in the order of 1.5 mg/g cell paste
(wet weight), which is slightly lower then the 3 mg/g cell paste
obtained for ISP
from B-356. The best preparations showed
two major bands on SDS-PAGE (Fig. 1). Their M
values were estimated to be 53,600 and 25,200, which correspond
to the M
values of the ISP
(plus the His tail addition) and
subunits, respectively. The M
of the native ISP
and His-tagged
ISP
were estimated by HPLC gel filtration and they were
found to be of 234,000 and 186,000, respectively. These values indicate
that the native conformation of B-356 ISP
is
and corroborate previously published
data obtained for strain LB400-ISP
(4) . The
lower native M
of His-tagged ISP
remains unexplained. However, the difference in migration in the
gel could be attributed to an electrostatic interaction between the His
tail and the diol residues of the stationary phase.
Figure 1:
SDS-PAGE of B-356
ISP subunits. Lane 1, M
markers; lane 2, preparation of B-356 ISP
(3 µg) obtained by the protocol described
previously(1) ; lane 3, Ni-NTA-purified preparation of
His-tagged B-356 ISP
(4 µg) obtained by PCR
amplification of bphAE; lane 4, Ni-NTA-purified
preparation of His-tagged B-356 ISP
subunit (2
µg) obtained by PCR amplification of bphA; lane
5, Ni-NTA-purified preparation of His-tagged B-356 ISP
subunit (2.5 µg) obtained by PCR amplification of bphE.
The purity of
different preparations of His-tagged ISP were evaluated
by scanning the SDS-PAGE gels, and these values were used as a
correction factor to evaluate the actual protein concentration of
His-tagged ISP
. Using these data, the average
value was calculated to be 8,300 M
cm
(based on the
determination on seven preparations). Purified His-tagged ISP
contained 2.6 ± 0.5 iron and 1.9 ± 0.1 sulfur per
mol of
/
heterodimer. Therefore, the number of iron atoms per
mol of protein for His-tagged ISP
preparations was close
to the theoretical value of 3 iron that should be expected for a
[2Fe-2S] Rieske-type center carrying a mononuclear
Fe
. As further evidence that the enzyme had retained
most of its mononuclear Fe
, we found that BPH dox
specific activity of His-tagged ISP
preparations
increased by only 30-40% when an excess of iron was added to the
reaction mixture.
On the other hand, the ISP preparations obtained from B-356 cells were somewhat more altered
than the His tail preparations. For example, based on the
value of 8,300 M
cm
, we calculated that on average, 85% of the
protein in the preparations had retained an intact Rieske center. This
estimation was confirmed by the observation that these ISP
preparations contained 1.7 ± 0.2 iron and 1.7 ± 0.3
sulfur atom/mol of
/
heterodimer. It thus appears that the
ISP
preparations obtained from strain B-356 had lost all
their mononuclear iron and that a portion of the Rieske center was
destroyed.
ISP remained active for months at -70
°C. However, the His-tagged ISP
was not as stable. We
observed that older preparations of His-tagged ISP
that
were reduced in activity showed a different subunit association pattern
where the preparation contained
monomer and
homodimer with
minor amounts of
. On the other hand,
heterodimer was the only form
observed in the case of B-356 ISP
. This suggested that
is the only active form of
ISP
.
We found that a 20-min preincubation of
His-tagged ISP with 5 mM dithiothreitol on ice
can restore activity of older preparations. The same phenomenon was
observed with FER
. However, it is not clear whether this
reactivation occurred by restoring the Rieske center or by changing
some other feature of the molecule. On the other hand, fresh
preparations were at their optimal level of activity when the required
supplemental mononuclear Fe
was added to the assay.
Addition of dithiothreitol did not significantly affect their activity.
Kinetic parameters of the recombinant versus parental
protein were compared when ISP in the reaction mixture
containing His-tagged RED
and His-tagged Fer
was replaced by His-tagged ISP
. For both
preparations, 40 °C was the optimal temperature for BPH dox
activity and the reaction was optimal at pH 5.5-6.0. When the
proportion of the enzyme components were varied in the assay, the
reaction was optimal for equimolar amounts of each one. Under the
conditions described under ``Experimental Procedures,'' the K
and V
for the BPH dox
activity were, respectively, 94 µM and 1.5 nmol
min
µg
for ISP
and 100 µM and 1.9 nmol min
µg
for His-tagged ISP
.
In
previous work, gas chromatography-mass spectroscopic analysis of
metabolites produced from B-356-BPH dox reaction suggested that both
2,3- and 3,4-dihydro-dihydroxybiphenyl were produced from
BPH(1) . Both metabolites were also produced when
B-356-ISP was replaced by the recombinant ISP
in the BPH dox assay (results not shown).
The
purified proteins were detected on SDS-PAGE (Fig. 1). Gel
filtration HPLC analysis showed that the native form of the purified
His-tagged subunit was a monomer (M
=
44,000), while the His-tagged
subunits had a tendency to join
together to produce homodimers plus large conglomerates. The UV visible
absorbance spectra of purified
subunit (but not
subunit)
was similar to the spectra of ISP
and His-tagged
ISP
(Fig. 2). There was a slight shift of the
peaks with maxima at 335 and 443 nm instead of 323 and 455 nm. After
reduction with excess dithionite under aerobic conditions, the spectra
resembled that of other reduced Rieske-type proteins. Assuming that the
of this protein is equal to the
of the ISP
complex, we have estimated that on
average 60% of the His-tagged
subunit of purified preparations
were carrying an intact Rieske center. This is supported by the
observation that the ratio of iron and acid-labile sulfur per mol of
His-tagged
subunit was 1.3 ± 0.4 iron and 1.3 ± 0.1
sulfur. Therefore, part of the Rieske center was either denatured
during purification of the His-tagged
subunit or was not formed
in the E. coli cells. The altered enzyme subunit was not
restored by adding dithiothreitol or iron. Contrary to the naphthalene
dioxygenase-
subunit(19) , ISP
-
subunit
showed a broad peak between 300 and 500 (Fig. 2B). This
peak was not modified under reduced conditions.
Figure 2:
A, absorption spectra of oxidized (solid line) His-tagged B-356 ISP (49
µM), oxidized (dotted line), and
dithionite-reduced (dashed line) His-tagged ISP
subunit (42 µM). B, absorption
spectra of ISP
subunit (55
µM).
When preparations of
individually purified subunits were combined in vitro and
tested in the BPH dox assay immediately or after 18 h of preincubation
at 4 °C, the activity recorded was only about 1% of the activity
obtained with His-tagged ISP preparation that had been
assembled in vivo. However, when the crude cell extracts were
mixed instead of the purified proteins, BPH dox activity was restored
to a higher level (Table 1). The activity increased with time
when the mixture was preincubated for 18 h at 4 °C (results not
shown) as it had been observed for the naphthalene
dioxygenase(18) . In order to find out which protein was
deactivated during the purification, we mixed a crude E. coli lysate containing one His-tagged subunit with a purified
preparation of the other His-tagged subunit. Using this approach, only
trace amounts of activity were obtained when purified
subunit was
added to the lysate but a fair level of activity was observed when
purified
subunit was used (Table 1). On the other hand,
when the crude cell extracts containing the individual subunits were
mixed and incubated for 18 h at 4 °C and then purified together on
Ni-nitrilotriacetic acid resin, the resulting His-tagged ISP
preparation was active (Table 1). These results suggested
that the
subunit was denatured when it was purified alone.
However, its prior association with the
subunit protects the
enzyme against deactivation. These data clearly showed that
and
subunits of ISP
can associate in vitro to
restore the active BPH dox.
In this work we have reported some of the major features of
strain B-356 His-tagged ISP and compared them to B-356
ISP
. Most of the data presented here, including the
arrangement of subunits are similar
to the recently published properties of LB400 oxygenase component. It
is not surprising because both enzymes are structurally closely related (11) . At this time, however, we cannot explain the significant
difference between the
value of 8,300 M
cm
we have determined
for B-356-ISP
and the one that was reported for strain
LB400 ISP
.
Our data show that the main features that
characterize B-356 ISP carrying a His tail on the
subunit are very similar to those found for the untagged parental
protein obtained from strain B-356 cells. Moreover, because the steps
required for purification are milder, the His-tagged enzyme component
preparations were less altered, as reflected by their UV visible
spectral data, the iron and acid labile sulfur content, and the
specific activity of the enzyme.
Hydroxylating dioxygenases are
multicomponent enzymes that catalyze the transfer of electrons from an
electron donor, usually a reduced nicotinamide dinucleotide, to the
hydroxylating center of the oxygenase component. Based on the number of
components and on the number and type of [2Fe-2S] centers
involved in electron transfer, hydroxylating oxygenases have been
subdivided into three classes(2, 3) . BPH dox belongs
to the class IIB dioxygenases which also includes benzene dioxygenase
and toluene dioxygenase. These enzyme systems comprise three
components, a reductase containing a flavin cofactor, a Rieske-type
ferredoxin protein, and a terminal oxygenase, which is a two-subunit
iron-sulfur protein containing a Rieske-type [2Fe-2S] center.
Absorbance spectra of purified B-356-BPH dox and LB400-BPH dox terminal
oxygenases were typical of Rieske-type
proteins(1, 4) . Moreover, sequence analysis has
identified a conserved sequence C-R-H-(aa)-C-S-Y-H- of a
Rieske center on the
subunit of both strains
ISP
(11, 20, 21) . However,
confirmation of the presence of the Rieske center on the
subunit
of class IIB oxygenases has never been established. Suen and Gibson (19) have obtained a purified preparation of the class III
naphthalene dioxygenase terminal oxygenase-
subunit. However,
their purified protein was denatured and did not show a typical
Rieske-type absorbance spectra. Therefore, use of His-tail protein
provided, for the first time, spectral evidence that the Rieske center
is located on the
subunit of the terminal oxygenase.
In spite
of the fact that approximately 60% of the ISP
subunit in purified preparations had retained an intact Rieske center
with [2Fe-2S], these preparations were only weakly active
when mixed with purified or crude preparations of
subunit.
Structural modifications during the purification process to prevent
reassociation with the
subunit cannot be excluded. This is also
supported by the observation that these preparations did not show
heteromers on gel filtration
(results not shown). The fact that the
subunit in crude cell
extracts can combine with exogenous
subunit to generate an active
complex might suggest the presence in cell extract of proteins or other
constituents that interact with the
subunit to protect their
folding. However, further study is needed to understand the reasons for
impaired activity.
The sequence of events that occurs at the active
site of aryl dioxygenases to bind and activate the molecular oxygen and
to attach it to the substrate is still unknown. The [2Fe-2S]
Rieske-type cluster found in the terminal oxygenase component is
presumed to involve at least two Cys and two His residues inside this
domain (2, 13) to coordinate the iron atoms. In
addition, other Tyr and His residues that are located farther away
inside the protein are presumed to be involved in the coordination of a
mononuclear Fe which is required for oxygen binding
to the enzyme(3, 13, 22) .
Batie et
al. (13) have clearly shown that Pseudomonas cepacia phthalate dioxygenase contains two iron atoms coordinated to Cys
and His residues. They also demonstrated that the presence of an
additional mononuclear Fe is required for enzyme
activity. Suen and Gibson (19) have also reported the presence
of 6 iron and 4 acid labile sulfide atoms per mol of purified Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase
oxygenase component, where this enzyme was found to be an
enzyme. Haddock and Gibson (4) have inferred the presence of a third mononuclear iron in
LB400-BPH dox terminal oxygenase from the observation that
Fe
stimulated the enzyme activity. Because of the
good quality of the B-356 His-tagged ISP
preparations,
our data provides evidence that this enzyme contains 3 iron atoms and 2
acid labile sulfur atoms per mol of
heterodimer.
It as
been suggested that the subunit of the aryl dioxygenase oxygenase
component is involved in substrate recognition. Hirose et al. (7) have used various hybrid clones of P. pseudoalcaligenes strain KF707-BPH dox genes and P. putida strain
F1-toluene dox genes, to show the importance of bphA2 (bphE) in determining substrate specificity. They made
the observation that bphA2 could not be replaced by todC2 to obtain functional BPH dox, but that bphA1 could be
replaced by todC1. Whatever the role of the
component,
it is certainly not carrying any Rieske center. This is confirmed by
sequence analysis of the gene (11) and from the UV visible
spectra of purified His-tagged ISP
subunit.
However, its association to the
subunit is essential to protect
the activity of the latter.
Our data clearly show that the assembly
of the ISP subunits can occur in vitro. We have
reported for the first time the purification of an in vitro assembled ISP terminal oxygenase. Because the recombinant
His-tagged components of the B-356-BPH dox have retained all properties
of the parental protein, including the activity, the QIAGEN
purification system appears, therefore, quite promising to further
explore the various features of bacterial dioxygenases. Particularly,
it opens the possibility of comparing reconstituted hybrid oxygenases
containing subunits belonging to terminal oxygenases of different
origin. Therefore, this system introduces a very useful new tool to
further investigate the major features that distinguish the various
aryl dioxygenase among them. Ongoing work in our laboratory is intended
to exploit this.