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J Biol Chem, Vol. 275, Issue 17, 12430-12437, April 28, 2000
From the The oxygenase component of biphenyl dioxygenase
(BPDO) from Comamonas testosteroni B-356 dihydroxylates
biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating
their degradation. Overexpressed, anaerobically purified BPDO had a
specific activity of 4.9 units/mg, and its oxygenase component appeared
to contain a full complement of Fe2S2 center
and catalytic iron. Oxygenase crystals in space group R3
were obtained under anaerobic conditions using polyethylene glycol as
the precipitant. X-ray diffraction was measured to 1.6 Å. Steady-state
kinetics assays demonstrated that BPDO had an apparent
kcat/Km for biphenyl of
(1.2 ± 0.1) × 106 M The microbial catabolic activities responsible for the degradation
of aromatic compounds constitute an essential link in the global carbon
cycle. These activities are of considerable practical interest due to
their potential to destroy toxic, persistent pollutants, a strategy
known as bioremediation (1). In the case of highly chlorinated,
structurally diverse xenobiotics such as
PCBs,1 the development of a
practical bioremediation technology has been limited in part by the
failure of existing microbial catabolic activities to effectively
degrade these compounds (1, 2). This failure may arise because these
activities have not yet evolved to degrade compounds that have only
recently been introduced into the biosphere. An important aspect of the
adaptation of catabolic activities for bioremediation is the study of
the structure and function of key catabolic enzymes. Such studies
provide insight into the molecular basis of an important biological
process, thereby facilitating the modification of enzyme specificity
and the design of novel metabolic pathways.
BPDO catalyzes the initial reaction in the aerobic degradation of
biphenyl and some PCBs. BPDO is a typical aromatic ring-hydroxylating dioxygenase, utilizing O2 and electrons originating from
NADH to transform biphenyl to
cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene (Fig. 1) (3, 4). This dihydroxylation
prepares the ring for subsequent degradation by ring cleavage enzymes.
The enzyme comprises an FAD-containing reductase (BphG), a Rieske-type
ferredoxin (BphF), and a two-subunit oxygenase of
BPDO is a major determinant of the PCB-catabolizing capabilities of
biphenyl-degrading strains, and the enzymes from different strains
possess significantly different congener-transforming abilities. For
example, BPDOLB400 from Burkholderia cepacia
LB400 transforms a much broader range of congeners than
BPDOKF707 from Pseudomonas pseudoalcaligenes
KF707, even though they share over 95% sequence identity.
Interestingly, BPDOKF707 transforms 4,4'-diClB much more
effectively than BPDOLB400 (7). BPDOB-356 from
Comamonas testosteroni B-356, which shares 70% sequence
identity with BPDOLB400 and BPDOKF707,
transforms a more limited range of congeners than BPDOLB400, but also transforms 4,4'-diClB poorly (8).
Moreover, BPDOB-356 and BPDOKF707 exclusively
catalyze the 2,3-dihydroxylation of biphenyl substrates (7, 9), whereas
the LB400 homologue catalyzes the 3,4-dihydroxylation of certain PCB
congeners (10). Finally, BPDOLB400 has been shown to
catalyze the dehalogenation of certain ortho-chlorinated
congeners (10). Significantly, no BPDO has been found to effectively
transform highly chlorinated biphenyls.
Several investigators have attempted to elucidate the structural
determinants of congener preference in BPDO with the ultimate objective
of enhancing this enzyme's PCB-transforming ability. Studies of
variant BPDOLB400 and BPDOKF707 implicate a
relatively small number of residues in the carboxyl terminus of the
oxygenase Detailed structural and biochemical information on BPDO would greatly
facilitate ongoing efforts to enhance this enzyme's PCB-degrading
capabilities and contribute to our understanding of ring-hydroxylating
dioxygenases. The ability to obtain such information has been limited
by the propensity of the BPDO oxygenase to form inclusion bodies in
Escherichia coli, its O2 lability, and the lack
of a continuous activity assay. We report here the improved expression
and purification of recombinant BPDOB-356. An oxygraph
assay was established to investigate the specificity of the enzyme and
its steady-state utilization of O2 in the presence of
different biphenyls. Conditions for crystallization of the BPDO
oxygenase were established, and analysis of preliminary x-ray diffraction data from these crystals is reported.
Materials--
Biphenyl was purchased from Aldrich (Mississauga,
Ontario, Canada) and Anachemia (Quebec, Canada). Catechol was purchased from Fisher (Quebec). 2,3-Dihydroxybiphenyl was a kind gift from Professor Victor Snieckus. 2,2'-diClB was purchased from Analabs, Inc.
(North Haven, CT). 3,3'- and 4,4'-diClBs were purchased from ULTRA
Scientific (North Kingstown, RI). Restriction enzymes and PfuI polymerase were purchased from Promega and Stratagene,
respectively. Synthetic oligonucleotides with the sequences
5'-CCGGATTAATTAGAGCTCCCGACACGTGC-3' and
5'-CAGGTGAAGGCCTTTGCGTTGCCA-3' were purchased from the Sheldon Biotechnology Center (McGill University, Montreal, Canada). The SacI restriction site introduced by the former is
underlined. Kits used to screen for potential crystallization
conditions were from Hampton Research (Laguna Niguel, CA), and PEGs
used in crystallization protocols were from Fluka (Milwaukee, WI).
Ferene S was purchased from ICN Biomedicals Inc. (Aurora, OH). All
other chemicals were of analytical or HPLC grade.
Bacterial Strains and Plasmids--
Strains used for protein
expression or DNA propagation included E. coli DH5 DNA Manipulation and Amplification--
DNA was purified using
the Wizard Plus Minipreps kit (Promega). DNA was digested, ligated, and
transformed into E. coli using standard protocols (24). DNA
was amplified using polymerase chain reactions containing 0.3 µg of
the template DNA, 2.5 units of Pfu polymerase, 0.4 mM each dNTP, and 20 pmol of each oligonucleotide primer in
a final volume of 25 µl. Twenty temperature cycles were performed
using a DNA thermal cycler (Perkin-Elmer) as follows: 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 45 s. Plasmids were
introduced into P. putida KT2442 by electroporation using the Pulse Controller and Gene Pulser from Bio-Rad (Ontario) with 1-mm
cuvettes (BTX, Inc., San Diego, CA) (25). DNA sequencing was performed
at the Sheldon Biotechnology Center using a Perkin-Elmer 373A sequencer
and the ABI sequencing strategy.
Protein Purification--
Chromatography was performed on an
ÄKTA Explorer (Amersham Pharmacia Biotech, Quebec). This system
was installed next to a Labmaster Model 100 glove box (M. Braun, Inc.
Peabody, MA) so that anaerobic buffers could be delivered to the former
and the column eluate could be directed to a fraction collector in the glove box (23). Buffers were prepared using water purified on a
Barnstead NANOpure UV apparatus to a resistivity of > 17.5 megaohms-cm. Buffer A contained 25 mM HEPES (pH 7.3), 10%
glycerol, 2 mM dithiothreitol, and 0.5 mM
ferrous ammonium sulfate. Buffer B was buffer A containing 1 M NaCl, and buffer PS was buffer A containing 5% saturated
ammonium sulfate. Buffers HA and HB contained 5 and 400 mM
sodium phosphate (pH 6.3), respectively, 10% glycerol, and 2 mM dithiothreitol. Warmed buffers were degassed with
N2 and equilibrated in the glove box for 24 h before
adding dithiothreitol and ferrous ammonium sulfate.
The washed cell pellet from 10 liters of culture was resuspended in 80 ml of 25 mM HEPES (pH 7.3) and 10% glycerol containing 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml DNase I, 1 mM MgCl2, and 2 mM
CaCl2. Cell suspensions were sonicated in 45-ml batches using a Virsonic 475 apparatus with 10 × 12-s pulses at level 6. The cell debris was removed by ultracentrifugation for 60 min at
300,000 × g. The clear supernatant was carefully
decanted, passed through a 0.45-µm filter, and diluted with 80 ml of
buffer A. This fraction, identified as crude extract, was degassed. All subsequent procedures were performed under an inert atmosphere unless
otherwise specified.
The crude extract was divided into five portions, each of which was
loaded onto a Source 15Q anion-exchange column (2 × 9 cm;
Amersham Pharmacia Biotech) equilibrated with buffer A. The column was
operated at a flow rate of 15 ml/min. Bound proteins were eluted with a
linear gradient of NaCl from 0 to 0.15 M over 10 column
volumes. Fractions with absorbances at 323 and 455 nm, characteristic
of the Rieske-type Fe2S2 center, and that
contained the expected polypeptides, as judged by SDS-polyacrylamide
gel electrophoresis, were concentrated to 20 ml by ultrafiltration using a stirred cell equipped with a YM-30 membrane (Amicon, Ontario) and filtered. The preparation was brought to 5% saturation with ammonium sulfate, divided into two equal portions, filtered, and loaded
onto a phenyl-Sepharose column (1 × 9 cm; Amersham Pharmacia Biotech) pre-equilibrated with buffer PS. The column was operated at a
flow rate of 0.75 ml/min. The oxygenase eluted at 0% saturated ammonium sulfate in a decreasing ammonium sulfate concentration gradient (5 to 0% over 2 column volumes). Reddish-colored fractions were concentrated by ultrafiltration and equilibrated with buffer HA by
gel filtration on a 0.7 × 12.5-cm column of Bio-Gel P6 DG (Bio-Rad). The sample was then loaded onto a hydroxylapatite type II
resin column (2 × 5 cm, 20-µm diameter; Bio-Rad), and the
protein was eluted with a linear gradient from 60 to 120 mM
sodium phosphate over 3 column volumes. Fractions of characteristic
absorbance were concentrated to 15-20 mg/ml by ultrafiltration and
flash-frozen as beads in liquid N2.
Analytical Methods--
SDS-polyacrylamide gel electrophoresis
with a 12% resolving gel was performed using a Bio-Rad MiniPROTEAN II
apparatus, and gels were stained with Coomassie Blue according to
established procedures (26). Protein concentrations were estimated
using the bicinchoninic acid protein assay reagent kit (Pierce) after removal of interfering substances (27). Bovine serum albumin was used
as a standard. Iron content was determined colorimetrically using
Ferene S (28). Sulfur content was determined colorimetrically with
N,N-dimethyl-p-phenylenediamine and
Na2S as a standard (29). Concentrations of recombinant
BphFLB400 and BphGB-356 were determined spectrophotometrically using Steady-state Kinetic Measurements--
Enzyme activity was
measured by following O2 consumption using a
computer-interfaced Clark-type polarographic O2 electrode (YSI Model 5301) (23) or a Hansatech DW1 O2 electrode with
a similar interface. Data were recorded every 0.1 s. Initial
velocities were determined from progress curves by analyzing the data
using Microsoft Excel. The slope of the progress curve and the
correlation coefficient of the slope were calculated for all
consecutive 6-s intervals using the full set of 61 data points
(23).
The standard activity assay contained 70 µM
Fe(SO4)2(NH4)2, 288 µM biphenyl, 123 µM NADH, 1.2 µM BphGB-356, 2.8 µM
BphFLB400, and 0.36 µM oxygenase. The
reaction was initiated by adding oxygenase after equilibrating the
assay with all other components for 20 s. The assay was performed
in a total volume of 1.3 ml of air-saturated 50 mM MES
(I = 0.05 M; pH 6.0) at 25.0 ± 0.1 °C. Buffers and stock solutions were prepared fresh daily. Stock
solutions and protein samples were prepared anaerobically, stored under
argon on ice, and withdrawn using a gas-tight syringe. The
O2 electrode was zeroed and calibrated on each day kinetic
assays were performed using either DHBD or, at O2
concentrations lower than 75 µM, catechol 2,3-dioxygenase
as described previously (23). Activity determinations were corrected
for the O2 consumption observed in the presence of NADH,
biphenyl, reductase, and ferredoxin only. One unit of enzyme activity
is defined as the quantity of enzyme required to consume 1 µmol of
O2/min under the standard assay conditions.
Apparent steady-state kinetic parameters for biphenyls were determined
by measuring rates of oxygen uptake in the presence of quantities of
biphenyls up to and exceeding their respective solubility limits. In
this respect, the solubilities of biphenyl and 2,2'-, 3,3'-, and
4,4'-diClBs were taken to be 45, 4.5, 1.6, and 0.27 µM,
respectively (32). These values, reported in water, are similar to
those in 50 mM MES (pH 6.0) at 25 °C as determined by
titrating buffer with stock solutions of biphenyls in ethanol and
monitoring UV-visible spectra for the appearance of turbidity at 400 nm
as well as the absorbance of the biphenyl at 250 nm.
For reactions performed using dissolved O2 concentrations
other than that of air-saturated buffer, reaction buffers were bubbled with appropriate mixtures of humidified O2 and
N2 gases for 15-30 min prior to the experiment as
described previously (23). The equilibrated buffer was transferred to
the reaction chamber using a gas-tight syringe, and the stopper was
inserted into the reaction chamber. The reaction chamber was flushed
continuously with the humidified gas mixture during this operation as
well as during the assay. For reactions performed using O2
concentrations lower than 2.5%, the reaction chamber was blanketed
with argon. Standard curves were established for the ranges 0-5,
5-20, and 20-100% O2. Initial velocities determined at
different substrate concentrations were fitted to the Michaelis-Menten
equation using the least-squares fitting and dynamic weighting options
of LEONORA (33).
Coupling Measurements--
Coupling experiments were carried out
in 50 mM MES (pH 6.0) at 25.0 ± 0.1 °C using
excess biphenyl substrate, 350 µM NADH, and the same
concentrations of BPDO components that were used in the standard
activity assay. Reactions were initiated by adding oxygenase and were
quenched 1-3 min later by diluting 300 µl of reaction mixture with
600 µl of methanol. Oxygen consumption was monitored using the
O2 electrode. The consumption of biphenyl substrate was
determined by HPLC measurements using a Hewlett-Packard 1050 Series
system equipped with a C18 reverse-phase column (0.46 × 15 cm; Higgins Analytical, Inc., Mountain View, CA). The instrument was operated with solvents under a constant helium purge and at a flow
rate of 1 ml/min. Biphenyl was eluted with a 20-ml gradient of 50%
H2O and 50% acetonitrile to 10% H2O and 90%
acetonitrile. Samples of 100 µl were injected, and the amount of
biphenyl was determined from the area of the absorbance peak at 203 nm
using a standard curve. Standard curves with correlation factors >0.95 were established by determining the peak areas of known amounts of
biphenyls. Samples for the standard curve were treated in the same way
as reaction mixtures to account for any losses of biphenyl incurred
during sample manipulation.
The amount of hydrogen peroxide produced during biphenyl transformation
was estimated using catalase, 650 units of which was added to the
reaction mixture at the time corresponding to the methanol quench. The
amount of O2 that was detected upon the addition of
catalase was taken to represent 50% of the hydrogen peroxide produced
during biphenyl transformation.
Crystallization of BPDO Oxygenase--
All crystallization
experiments were performed under anaerobic conditions in a
N2 atmosphere glove box (Innovative Technologies, Newburyport, MA) maintained at X-ray Data Collection and Analysis--
All diffraction patterns
were recorded from frozen crystals by the rotation/oscillation method.
The patterns were analyzed and reduced to average intensities by the
use of the HKL program suite (35); intensities were converted to
structure factor amplitudes using programs from the CCP4 package (36).
Crystals were prepared for diffraction experiments by soaking them for
1 min in 400 µl of a solution containing 100 mM sodium
citrate (pH 5.8), 10% (v/v) 2-propanol, 24% (w/v) PEG 4000, and 20%
(v/v) glycerol before flash-freezing by direct immersion in liquid
N2. Initial, partial diffraction patterns were obtained by
the use of CuK Construction and Testing of the Expression Vector--
Digestion
of pUCBPHA-C (21) with KpnI yielded a 3178-bp fragment that,
according to the sequence (37), contains the genes encoding
BPDOB-356 oxygenase (bphAE) together with 1 kilobase pair of flanking upstream DNA. This fragment was purified and ligated with KpnI-digested and dephosphorylated pVLT31, a
broad host range expression vector (20). The construct containing the
cloned fragment in the correct orientation, as verified by restriction
digestion using SmaI, was designated pVLT31AE7. P. putida KT2442 containing pVLT31AE7 failed to express significant levels of BPDO when induced (data not shown).
A second construct (designated pVLT31AE7-3) was derived from pVLT31AE7
by removing ~1 kilobase pair between the Ptac promoter of
pVLT31 and the ribosome-binding site of bphA. Briefly, a
411-bp fragment of DNA containing the 5'-end of bphA and its ribosome-binding site was amplified by polymerase chain reaction using
the oligonucleotides described under "Experimental Procedures," purified from an agarose gel, and digested with SacI and
SacII. This fragment was ligated into pVLT31AE7 digested
with SacI and SacII, thereby replacing the
1400-bp SacI-SacII fragment of pVLT31AE7 with a
351-bp SacI-SacII fragment. In pVLT31AE7-3, the
Ptac promoter is located 71 bp upstream of the ATG start codon
of bphA. This construct was partially sequenced to ensure
the absence of polymerase chain reaction-induced errors. P. putida KT2442 containing pVLT31AE7-3 expressed high levels of
soluble BPDO under appropriate induction conditions. Attempts to
improve this expression by modifying the ribosome-binding site upstream
of bphA were not successful (data not shown).
Purification of BPDO--
The relevant details of the anaerobic
purification of BPDO from P. putida KT2442 containing
pVLT31AE7-3, which was completed over a 20-h period, are summarized in
Table I. SDS-polyacrylamide gel
electrophoresis followed by Coomassie Blue staining revealed that the
oxygenase was of comparable purity to the aerobically purified
preparation (3). The specific activity of purified BPDO was 4.9 units/mg. The iron content of the purified oxygenase was estimated to
be from 8.5 to 12.0 mol/mol of BPDO by the chemical Ferene S iron assay
for different preparations. The sulfur content was estimated to be 5.3 mol/mol of oxygenase hexamer. Under aerobic conditions, the absorbance
spectrum was similar to that of BPDO purified aerobically from C. testosteroni strain B-356 (3) and was characteristic of a
Rieske-type Fe2S2 cluster with maxima at 323 and 455 nm and a shoulder at ~575 nm. In the oxidized form of BPDO
oxygenase, the ratio of the absorbance maxima at 280 and 323 nm was
9.2.
Oxygen Uptake Assays--
In the presence of biphenyl,
O2 uptake rates were dependent on the concentrations of
BphFLB400 and BphGB-356. In the presence of
0.36 µM oxygenase, the initial rate of O2
uptake was similar at ratios of 3 and 6 molecules of
BphG/ Steady-state Kinetic Analysis--
Under the standard assay
conditions, BPDO exhibited Michaelis-Menten kinetics for the dependence
of the initial rate of O2 uptake on the concentration of
biphenyl (Fig. 2A). The
biphenyl concentration could be varied sufficiently within its
solubility range to allow reliable estimates of the apparent
kcat and Km (Table
II). The initial rates of O2
uptake by BPDO in the presence of 2,2'- and 3,3'-diClBs, respectively,
also fit the Michaelis-Menten equation very well, and random trends in
the residuals were observed (data not shown). Interestingly, the
maximum rates observed in the presence of these compounds were observed
at biphenyl concentrations that apparently exceeded their respective
solubility limits. This is particularly striking for 3,3'-diClB, for
which the apparent Km is approximately twice its
solubility limit (Table II). The steady-state parameters of BPDO for
4,4'-diClB could not be estimated due to the extremely limited aqueous
solubility of this compound (0.27 µM) and the low initial
rates of O2 uptake. Thus, for this diClB, only the maximum
initial rate of O2 uptake observed is reported in Table
II.
BPDO also exhibited Michaelis-Menten behavior for the dependence of the
initial rate of O2 uptake on the concentration of O2 in the presence of saturating amounts of biphenyl (Fig.
2B) as well as 2,2'- and 3,3'-diClBs (data not shown).
Interestingly, the apparent
kcat/Km of BPDO for
O2 in the presence of biphenyl was 10- and 50-fold higher
than in the presence of 3,3'- and 2,2'-diClBs, respectively (Table
III). In the presence of 4,4'-diClB, the
initial rate of O2 uptake increased linearly with the
concentration of O2, indicative of a very high
Km.
In an attempt to elucidate the steady-state kinetic mechanism of BPDO,
rate data obtained at different concentrations of biphenyl (5-100
µM) and O2 (15-250 µM) were
fitted to ternary complex and substituted enzyme mechanisms (data not
shown). Both fits yielded kinetic parameters similar to those reported
above, with standard errors ranging from 4 to 15%. Comparison of the
fits using the F test indicated that the quality of the fits was not
significantly different. Moreover, product inhibition was not available
as a kinetic tool since O2 consumption was observed in the
presence of
cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene
(data not shown). Therefore, the kinetic mechanism could not be
determined using either approach.
To determine whether substrate utilization by BPDO is coupled, the
stoichiometry of biphenyl substrate and O2 consumed in the
enzyme-catalyzed reaction was investigated. In the presence of a
saturating concentration of biphenyl (125 µM), the amount of O2 consumed corresponded to the amount of biphenyl
utilized, within experimental error (Table II). Furthermore, no
hydrogen peroxide, a possible uncoupling product, was detected upon the addition of catalase to the O2 electrode chamber. In
contrast, the utilization of diClB and O2 by BPDO was
significantly uncoupled: the amounts of 2,2'-, 3,3'-, and 4,4'-diClBs
transformed corresponded to ~50% of the O2 consumed.
Within experimental error, the balance of consumed O2 was
detected as hydrogen peroxide (Table II).
Crystallization Trials--
Several experiments in the initial
crystallization screens produced brown crystallites. The productive
precipitants included PEG 4000, PEG 8000, ammonium sulfate, and
2-propanol. Variation of the buffer and pH as well as the
concentrations of 2-propanol and PEG 4000 rapidly yielded crystals
suitable for diffraction experiments. Characteristically brown crystals
with a rhombic morphology grew in 1-2 weeks to a typical size of
0.3 × 0.1 × 0.1 mm.
Diffraction Studies--
Analysis of diffraction patterns
established that the crystals belonged to the space group R3
with unit cell parameters for the hexagonal setting of
a = b = 136.35 Å and c = 106.07 Å (Fig. 3). Test images
collected by the use of a rotating anode source showed diffraction to
2.2-Å resolution and a nominal mosaicity of 0.60°. The complete
diffraction pattern was subsequently measured using synchrotron
radiation to 1.6-Å resolution with a refined mosaicity of 0.46°.
Scaling yielded an overall Rsym of 8.7%;
statistics as a function of resolution are presented in Table
IV. Analysis of the cumulative intensity
distribution with the CCP4 program TRUNCATE (38) revealed
that the crystal was twinned. Further evaluation of the data by the use
of a Web-based twinning analysis package (39) revealed merohedral
twinning about the hexagonal a/b axes and a twinning
fraction of 0.34. The data used to prepare Table IV were not corrected
for twinning.
The described overexpression and rapid, anaerobic purification of
BPDOB-356 oxygenase reproducibly yielded 55 mg of highly active enzyme from 10 liters of cell culture. The specific activity of
the preparation was significantly greater than that reported for
aerobic preparations of the enzyme (4, 40), although direct comparisons
are difficult as the activity assay in the current study was optimized.
Nevertheless, iron and sulfur analyses are consistent with the presence
of a full complement of Rieske-type Fe2S2
cluster and mononuclear iron in the purified oxygenase based on
theoretical values of three iron and two sulfur atoms/ Although BPDO is a major determinant of the aerobic microbial
degradation of PCBs, many aspects of its reactivity with different congeners remain unknown. Most studies of the ability of BPDO to
transform different PCBs are based on single time point assays and have
focused on the identity of the dioxygenation products (e.g.
Ref. 41). Many of these were performed using whole cells and/or
mixtures of PCB congeners. Activity assays have been limited by the
multicomponent nature of BPDO, its O2 lability, and the limited solubilities of chlorinated biphenyls. The continuous activity
assay described herein overcomes many of these limitations, enabling a
more thorough characterization of the reactivity of BPDO toward
chlorinated biphenyls. Significantly, BPDO activity could not be
saturated with the ferredoxin. Similarly, the oxygenase of phthalate
dioxygenase, a related two-component enzyme, could not be saturated
with its reductase (42). Moreover, we were unable to determine the
steady-state kinetic mechanism of BPDO using the improved assay,
although previous studies of ring-hydroxylating dioxygenases have
established that these enzymes utilize a mechanism in which binding of
the aromatic substrate precedes O2 binding (44-46). The
determined apparent kinetic parameters nevertheless provide valuable
insights into the specificity of BPDO.
The steady-state kinetic parameters for BPDO indicate that the
Km for biphenyl is well below the solubility limit of this substrate and that the turnover number is low. The
Km(O2) of 28 µM indicates that BPDO is close to saturation with
O2 under ambient conditions. In contrast, DHBD, the
extradiol dioxygenase of the bph pathway, has a
Km(O2) of 1.3 mM (20 mM HEPPS and 80 mM NaCl (pH
8) at 25 °C) (23), although these two dioxygenases presumably
function under similar cytoplasmic conditions, including O2
concentrations. However, the
kcat/Km(O2)
of DHBD is an order of magnitude greater than that of BPDO, and even in
air-saturated buffer, the apparent specificity of DHBD for its aromatic
substrate is an order of magnitude greater than that of BPDO. Ongoing
studies in our laboratories indicate that these differences cannot be attributed to the different experimental conditions. Moreover, the
apparent physiological
kcat/Km(O2)
of BPDO is probably even lower, as the ratio of BphF to oxygenase is
much lower in vivo than in the current assay.
The maximum rates of O2 utilization by BPDO observed in the
presence of different diClBs are consistent with the ability of whole
cells of C. testosteroni B-356 (8) and purified His-tagged BPDOB-356 (9) to transform diClBs determined using single
time point assays. Accordingly, it was reported that 3,3'-diClB is transformed at rates ~5 times faster than either 2,2'- or 4,4'-diClB. In the current study, it was found that the maximum rate of
O2 consumption by BPDO in the presence of 3,3'-diClB was
~3.5 times higher than that in the presence of 2,2'-diClB (Table II).
Moreover, the consumption of O2 was 50% better coupled to
biphenyl transformation in the presence of 3,3'-diClB compared with
2,2'-diClB. However, it is not clear why the
Vmax of BPDO in the presence of diClBs was
attained at levels of these compounds that exceeded their solubility
limits or whether the enzyme is saturated with these compounds at the
observed Vmax values. The attainment of
Vmax at such high levels of diClBs could be due
to partitioning of the substrate to the active site of the enzyme from
the solid phase. Regardless of these considerations, it is interesting
to note that although 2,2'-diClB is more soluble than 3,3'-diClB, it is
transformed much less efficiently, suggesting that 3,3'-diClB is a
better substrate for BPDO.
The current data demonstrate that chloro substituents not only
influence the specificity of BPDO, but also dramatically affect its
ability to utilize O2. The ability of BPDO to utilize
O2 is reflected in two parameters: the
kcat/Km(O2)
and the coupling of biphenyl transformation to O2
consumption. Although many details of the catalytic mechanism of
ring-hydroxylating dioxygenases have yet to be established, most
studies are consistent with a mechanism whose initial steps follow the
cytochrome P450 paradigm (6, 43). Accordingly, the resting-state
dioxygenase first binds the aromatic substrate. Spectroscopic studies
in phthalate dioxygenase indicate that this binding converts the
high-spin hexacoordinate active-site Fe(II) to a pentacoordinate state
(44-46). The pentacoordinate Fe(II) then binds O2, and the
catalytic center is reduced to yield an activated oxygen species that
dioxygenates the aromatic substrate. Chlorinated biphenyls that do not
occupy the active site of BPDO in the same manner as biphenyl may not efficiently convert the hexacoordinate center to a pentacoordinate state, perhaps through failure to adequately displace active-site solvent species. This is analogous to the failure of some substrates to
convert low-spin Fe(III) to high-spin Fe(III) in cytochrome P450
(e.g. Ref. 47). Alternatively, chloro substituents might occlude the O2-binding site. Either effect would decrease
the kcat/Km(O2),
as observed. The diClB-induced uncoupling in BPDO is also analogous to
the cytochrome P450 paradigm. In cytochrome P450cam, poorly
coupled camphor analogues fail to displace active-site solvent species,
presumably allowing protons to react inappropriately with an activated
oxygen species (48). The diClB-induced production of
H2O2 in BPDO may arise from the same
phenomenon. Consistent with this reasoning, uncoupling in naphthalene
dioxygenase is induced by benzene (30), which is much smaller than the
enzyme's preferred substrate. Further study of uncoupling in BPDO is
warranted in light of its ramifications for PCB degradation.
The transformation products of diClBs obtained using
BPDOB-356 were not examined in this study. However,
products of diClBs generated using a His-tagged preparation of
BPDOB-356 have been partially characterized by gas
chromatography-mass spectrometry (9). In several cases, the low yields
of transformation products precluded their unambiguous identification
and the determination of their relative amounts. The transformation of
2,2'-diClB yielded two products: a monochlorinated dihydroxybiphenyl
and a dichlorinated dihydrodiol.4 The
monochlorinated dihydroxybiphenyl had gas chromatography-mass spectrometry spectra that were similar to those of
2,3-dihydroxy-2'-chlorobiphenyl, formed by BPDOLB400 (10).
The dichlorinated dihydrodiol differed from that formed by
BPDOLB400, which was also not unambiguously identified
(10). The transformation of 3,3'-diClB yielded a single dichlorinated
dihydrodiol that can be transformed by the following two enzymes of the
biphenyl degradation pathway. This compound was thus identified as
2,3-dihydro-2,3-dihydroxy-3',5-diClB (9). Finally,
BPDOB-356 transformed 4,4'-diClB to two products, one of
which was tentatively identified as
2,3-dihydro-2,3-dihydroxy-4,4'-diClB.4 The more active
preparation of BPDO reported herein should facilitate a more
comprehensive characterization of these transformation products.
The reactivity of BPDO with diClBs has several important implications
for the microbial degradation of PCBs, which are generally present in
the environment as complex mixtures of congeners. First, congeners such
as 2,2'-diClB that bind to the active site of BPDO oxygenase but that
are poorly transformed should competitively inhibit the transformation
of other congeners. Second, the uncoupling observed in the presence of
diClBs demonstrates that the degradation of some congeners exacts a
high energetic cost from the cell, since reducing equivalents are
transferred to O2. Moreover, reactive oxygen species that
result from this uncoupling may be deleterious to the cell. Such
uncoupling may explain in part why natural isolates are unable to
utilize biphenyls that are chlorinated on both phenyl rings as growth
substrates. Further study of the kinetics of PCB transformation
combined with the emerging structural data of BPDO oxygenase and
mutagenesis studies should provide critical insight into the molecular
basis of substrate specificity and substrate coupling in this enzyme.
We thank Nathalie M. Drouin for skilled
technical assistance. Manon M. J. Couture (Department of
Biochemistry, Université Laval) and Roch Aumont (Department of
Chemistry and Biochemistry, Concordia University) generously provided
purified BphFLB400 and BphGB-356, respectively.
Professor Michel Sylvestre (Institut National de la Recherche
Scientifique-Sante, Université du Québec) generously
provided pUCBPHA-C and His-tagged BphFB-356. Frederic H. Vaillancourt and Dr. Stephen Y. K. Seah (Department of
Biochemistry, Université Laval) generously provided DHBD and
catechol 2,3-dioxygenase, respectively. We thank Mathew N. Chakko
(Purdue University) for assistance in the crystallization experiments.
*
This work was supported in part by Natural Sciences and
Engineering Research Council of Canada Strategic Grant STP0193182 (to
L. D. E. and J. B. P.) and by National Institutes of Health Grant
GM-52381 (to J. T. B.). Use of the Advanced Photon Source was
supported by the United States Department of Energy, Basic Energy
Sciences, Office of Energy Research, under Contract W-31-109-Eng-38. BioCARS Sector 14 was supported by National Institutes of Health National Center for Research Resources Grant RR-07707.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a Natural Sciences and Engineering Research Council of
Canada postgraduate scholarship.
¶
To whom correspondence should be addressed: Dept. of Chemistry
and Biochemistry, Concordia University, 1455 de Maisonneuve Blvd., W.,
Montreal, Quebec H3G 1M8, Canada. Tel.: 514-848-8727; Fax:
514-848-2868; E-mail: Powlow@vax2.concordia.ca.
2
M. M. J. Couture, E. Babini, C. L. Colbert, J. T. Bolin, and L. D. Eltis, manuscript in preparation.
3
R. Aumont, personal communication.
4
M. Sylvestre, personal communication.
The abbreviations used are:
PCBs, polychlorinated biphenyls;
BPDO, biphenyl dioxygenase;
diClB, dichlorobiphenyl;
PEG, polyethylene glycol;
HPLC, high performance
liquid chromatography;
DHBD, 2,3-dihydroxybiphenyl dioxygenase;
MES, 2-(N-morpholino)ethanesulfonic acid;
bp, base pair(s);
HEPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic
acid.
Steady-state Kinetic Characterization and Crystallization of a
Polychlorinated Biphenyl-transforming Dioxygenase*
§,¶,
,, and
Department of Chemistry and Biochemistry,
Concordia University, Montreal, Quebec H3G 1M8, Canada, the
Department of Biological Sciences, Purdue University, West
Lafayette, Indiana 47907-1392, and the ** Department of Biochemistry,
Université Laval, Quebec City, Quebec G1K 7P4, Canada
ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1
s1 in air-saturated buffer. Moreover, BPDO transformed
dichlorobiphenyls (diClBs) in the following order of apparent
specificities: 3,3'- > 2,2'- > 4,4'-diClB. Strikingly, the ability of
BPDO to utilize O2 depended strongly on the biphenyl
substrate:
kcat/Km(O2) = (3.6 ± 0.3), (0.06 ± 0.02), and (0.4 ± 0.07) × 105 M1 s1 in the
presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover,
biphenyl/O2 consumed was 0.97, 0.44, 0.63, and 0.48 in the
presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively.
Within experimental error, the balance of consumed O2 was
detected as H2O2. Thus, PCB congeners such as
2,2'-diClB exact a high energetic cost, produce a cytotoxic compound
(H2O2), and can inhibit degradation of other
congeners. Each of these effects would be predicted to inhibit the
aerobic microbial catabolism of PCBs.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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3
3 constitution that contains a
Rieske-type Fe2S2 cluster and a mononuclear
iron center. Accordingly, BPDO has been classified as a group IIB
aromatic ring-hydroxylating dioxygenase together with benzene and
toluene dioxygenases (5). Structural and spectroscopic studies of
related dioxygenases indicate that the mononuclear iron center
orchestrates substrate transformation (reviewed in Ref. 6). BphG, BphF, and the oxygenase Fe2S2 cluster function to
transfer electrons from NADH to this center.
View larger version (14K):
[in a new window]
Fig. 1.
BPDO of C. testosteroni
B-356. The enzyme comprises an FAD-containing reductase
(BphG), a Rieske-type ferredoxin (BphF), and an oxygenase that contains
a Rieske-type Fe2S2 cluster and a catalytic
mononuclear iron center. Biphenyl is stereospecifically hydroxylated at
positions 2 and 3, yielding
cis-(2R,3S)-dihydroxy-2,3-dihydrobiphenyl.
ox, oxidized; red, reduced.
-subunit in determining congener preference (11, 12).
Moreover, chimeric oxygenases obtained by shuffling the genes encoding
BPDOLB400 and BPDOKF707 possess enhanced
PCB-degrading abilities with respect to the parental enzymes (13, 14).
This is consistent with studies of related enzymes, such as naphthalene
dioxygenase, in which the determinants of substrate specificity appear
to reside in the carboxyl terminus of the -subunit (15). However,
the substrate preferences of hybrid oxygenases in which the - and -subunits originate from more divergent enzymes, such as
BPDOLB400, BPDOB-356, and BPDOP6,
clearly indicate that the -subunit contributes to determining
substrate preference (9, 16).
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(17),
E. coli JM109 (18), and Pseudomonas putida KT2442
(19). E. coli and P. putida were grown at 37 and 30 °C, respectively. Plasmids used were pVLT31 (20) and pUCBPHA-C, a
derivative of pUC18 that contains a 6.3-kilobase pair
SmaI-SmaI fragment of C. testosteroni
B-356 DNA that includes bphAEXFBC (21). Strains harboring
pVLT31 and its derivatives were grown in the presence of tetracycline
(10 µg/ml). For BPDO expression, strains were grown in Luria-Bertani
broth containing a phosphate buffer originally described for Terrific
Broth (22) and supplemented (10 ml/liter) with an HCl-solubilized
solution of minerals (23). One liter of medium in a 2.8-liter Fernbach
flask was inoculated with 9 ml of an overnight culture. When the
A600 of the culture reached 0.7, isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 1 mM, and the culture was incubated for an additional 18 h before harvesting by centrifugation. The harvested cell pellet was washed twice with 500 ml of 25 mM
HEPES (pH 7.3) containing 10% glycerol and frozen until use.
326 = 9 mM1
cm1 2 and
450 = 11.8 mM1
cm1,3
respectively. 2,3-Dihydroxybiphenyl dioxygenase (DHBD) and catechol 2,3-dioxygenase were prepared as described previously (23, 31).
2 ppm O2. Samples of BPDO
were prepared and stored under liquid N2 as described
above. A typical sample contained 20 mg/ml enzyme, 80 mM
sodium phosphate (pH 6.3), 10% (v/v) glycerol, and 2 mM
dithiothreitol. The sitting-drop vapor-diffusion technique was used for
all experiments. At initial conditions, the sitting drop had a volume
of 4 µl and contained the protein sample and the well solution in a
1:1 ratio. The well volume was 1 ml. The sparse matrix method (34), as
incorporated within the Hampton Research Crystal Screen I kit, was used
at 20 and 10 °C to search for preliminary crystallization
parameters. Improvement of promising conditions was pursued by
variation of two parameters over a 24-cell grid. Crystals used in the
diffraction experiments described below grew at 20 °C. The well
solution initially contained 100 mM sodium citrate (pH
5.8), 10% (v/v) 2-propanol, and 24% (w/v) PEG 4000.
radiation from a Rigaku rotating anode
x-ray generator operated at ~50 kV and 100 mA and equipped with
focusing mirror optics and an R-axis IV imaging plate area detector
(Molecular Structures Corp.). A cryogenic crystal-cooling device
(Oxford Cryosystems, Oxford, United Kingdom) was used to maintain a
nominal sample temperature of 110 K. Crystals were recovered after the
initial experiments and stored under liquid N2 until
diffraction studies were resumed at the Advanced Photon Source
synchrotron using BioCARS beam line BM14D. At BM14D, diffraction
patterns were measured at a nominal sample temperature of 108 K
(Cryojet cooling device, Oxford Instruments USA, Boston, MA) using
monochromatic x-rays of 1-Å wavelength, a crystal-to-detector distance
of 74 mm, and a Quantum-1 CCD detector (Area Detector Systems Corp.,
Poway, CA).
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
Purification of the oxygenase component of the biphenyl dioxygenase
system from C. testosteroni B-356
33-hexamer, but in the latter case,
the background rate of O2 uptake was considerably higher.
With respect to BphF, the initial rate of O2 uptake was not
saturated even at 20 molecules of BphF/oxygenase -subunit, the
highest ratio tested. Significantly, BPDO activities obtained using
BphFLB400 and histidine-tagged BphFB-356 were
identical within experimental error. To standardize the activity assay, the concentrations of each dioxygenase component were 0.36 µM oxygenase -subunit, 2.8 µM
BphFLB400, and 1.2 µM BphGB-356
in all subsequent experiments. Under these conditions, the reductase concentration was close to saturating, and the background rate of
O2 uptake was <10% of that in the presence of saturating
concentrations of biphenyl.
View larger version (16K):
[in a new window]
Fig. 2.
Steady-state dihydroxylation of biphenyl by
BPDOB-356. A, the dependence of the initial
velocity of O2 uptake on biphenyl concentration in
air-saturated buffer. The fitted parameters were Km = 6.2 ± 0.5 µM and Vmax = 141 ± 3 µM min 1. B, the
dependence of the initial velocity of O2 uptake on
O2 concentration in the presence of a nominal biphenyl
concentration of 288 µM. The fitted parameters were
Km(O2) = 28 ± 2 µM and Vmax = 227 ± 9 µM min1. All experiments were performed
using 0.36 µM oxygenase and 50 mM MES (pH
6.0) at 25 °C. Solid lines represent fits of the data to
the Michaelis-Menten equation obtained using the least-squares dynamic
weighting options of LEONORA. The two highest concentrations of
biphenyl in A are above the published aqueous solubility
limit.
Apparent steady-state kinetic and coupling parameters of BPDO from C. testosteroni B-356 for selected biphenyl substrates
The apparent steady-state kinetic utilization of O2 by BPDO
from C. testosteroni B-356 in the presence of selected biphenyl
substrates
View larger version (115K):
[in a new window]
Fig. 3.
High-resolution diffraction pattern obtained
from a single crystal of BPDO oxygenase. The x-ray source was
synchrotron radiation as described under "Experimental Procedures."
The image was recorded for 45 s over an oscillation angle of 1°
at a crystal-to-detector distance of 73.8 mm. The edge of the image
corresponds to a resolution of 1.75 Å.
Statistics pertaining to diffraction data acquired from one crystal of
C. testosteroni B-356 BPDO
/
is the
average intensity divided by the average error as defined by the
HKL programs.
is the mean intensity of the j
observations of a reflection with reduced Miller indices
hkl.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit. In
contrast, aerobically purified preparations of BPDO oxygenase contained
less than a full complement of iron (3, 4). Moreover, stability
problems have been noted in a His-tagged form of the oxygenase
expressed in E. coli (40). The improved overexpression and
purification described herein overcome some significant shortcomings of
previously reported procedures, facilitating careful biochemical study
of the enzyme.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-0042; Fax: 604-822-6041; E-mail: leltis@interchange.ubc.ca.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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E. R. Goncalves, H. Hara, D. Miyazawa, J. E. Davies, L. D. Eltis, and W. W. Mohn Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1 Appl. Envir. Microbiol., September 1, 2006; 72(9): 6183 - 6193. [Abstract] [Full Text] [PDF]
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Y. Jouanneau and C. Meyer Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols. Appl. Envir. Microbiol., July 1, 2006; 72(7): 4726 - 4734. [Abstract] [Full Text] [PDF]
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D. Barriault and M. Sylvestre Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III J. Biol. Chem., November 12, 2004; 279(46): 47480 - 47488. [Abstract] [Full Text] [PDF]
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K. Furukawa, H. Suenaga, and M. Goto Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution J. Bacteriol., August 15, 2004; 186(16): 5189 - 5196. [Full Text] [PDF]
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A. Okuta, K. Ohnishi, and S. Harayama Construction of Chimeric Catechol 2,3-Dioxygenase Exhibiting Improved Activity against the Suicide Inhibitor 4-Methylcatechol Appl. Envir. Microbiol., March 1, 2004; 70(3): 1804 - 1810. [Abstract] [Full Text] [PDF]
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M. Sondossi, D. Barriault, and M. Sylvestre Metabolism of 2,2'- and 3,3'-Dihydroxybiphenyl by the Biphenyl Catabolic Pathway of Comamonas testosteroni B-356 Appl. Envir. Microbiol., January 1, 2004; 70(1): 174 - 181. [Abstract] [Full Text] [PDF]
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R. E. Parales, N. C. Bruce, A. Schmid, and L. P. Wackett Biodegradation, Biotransformation, and Biocatalysis (B3) Appl. Envir. Microbiol., October 1, 2002; 68(10): 4699 - 4709. [Full Text] [PDF]
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Y. Ge, F. H. Vaillancourt, N. Y. R. Agar, and L. D. Eltis Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen J. Bacteriol., August 1, 2002; 184(15): 4096 - 4103. [Abstract] [Full Text] [PDF]
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M. E.-S. Mohamed, A. Zaar, C. Ebenau-Jehle, and G. Fuchs Reinvestigation of a New Type of Aerobic Benzoate Metabolism in the Proteobacterium Azoarcus evansii J. Bacteriol., March 15, 2001; 183(6): 1899 - 1908. [Abstract] [Full Text]
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