Determination of the metal ion dependence and substrate
specificity of a hydratase involved in the degradation
pathway of biphenyl/chlorobiphenyl
Pan Wang and Stephen Y. K. Seah
Department of Microbiology, University of Guelph, Ontario, Canada
Microbial degradation of aromatic compounds is
important for maintaining the global carbon cycle and
also for the bioremediation of man-made aromatic
compounds, released in the environment due to industrial
activities. Diverse aromatic compounds, including
biphenyl [1], xylenes [2] and 3-(3-hydroxyphenyl)propionate
[3], can be degraded via the meta-cleavage
pathway, where the respective compounds is first converted
to a catechol. Subsequent enzymatic transformation
of the cleaved catechol leads to the formation
of the common intermediate 2-hydroxypent-2,4-dienoate
(HPDA). This intermediate can then be converted
to pyruvate and acetyl CoA by three further steps in
the pathway. The first of these three steps is catalyzed
by a divalent cation-dependent hydratase (EC 4.2.1.80)
that transforms HPDA to 2-hydroxy-4-ketopentanoate
(Fig. 1).
Among the meta-cleavage pathway enzymes, the last
three enzymes, responsible for the final formation of tricarboxylic
acid cycle intermediates, are comparatively
Keywords
aromatics; hydratase; 2-hydroxypent-2,4dienoate;
metal cofactor; substrate
specificity
Correspondence
S. Y. K. Seah, Department of Microbiology,
University of Guelph, Guelph, Ontario,
Canada, N1G 2W1
Fax: +1 519 837 1802
Tel: +1 519 824 4120 Ext. 56750
E-mail: sseah@uoguelph.ca
(Received 22 October 2004, revised 9
December 2004, accepted 15 December
2004)
doi:10.1111/j.1742-4658.2004.04530.x
BphH is a divalent metal ion-dependent hydratase that catalyzes the formation
of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate
(HPDA). This reaction lies on the catabolic pathway of numerous aromatics,
including the significant environmental pollutant, polychlorinated
biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia
xenoverans LB400, was overexpressed and purified to homogeneity. Atomic
absorption spectroscopy and Scatchard analysis reveal that only one
divalent metal ion is bound to each enzyme subunit. The enzyme exhibits
the highest activity when Mg2+
was used as cofactor. Other divalent
cations activate the enzyme in the following order of effectiveness:
Mg2+
> Mn2+
> Co2+
> Zn2+
> Ca2+
. This differs from the metal
activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy
of the Co2+
­BphH complex indicates that the divalent metal ion is
hexa-coordinated in the enzyme. The nature of the metal ion affected only
the kcat and not the Km values in the BphH hydration of HPDA, suggesting
that cation has a catalytic rather than just a substrate binding role.
BphH is able to transform alternative substrates substituted with methyland
chlorine groups at the 5-position of HPDA. The specificity constants
(kcat / Km) for 5-methyl and 5-chloro substrates are, however, lowered by
eight- and 67-fold compared with the unsubstituted substrate. Significantly,
kcat for the chloro-substituted substrate is eightfold lower compared with
the methyl-substituted substrate, showing that electron withdrawing substituent
at the 5-position of the substrate has a negative influence on enzyme
catalysis.
Abbreviations
BphH, MhpD, XylJ, HPDA hydratases; HPDA, 2-hydroxypent-2,4-dienoate; HODA, 2-hydroxy-6-oxohexa-2,4-dienoate; PCBs, polychlorinated
biphenyls; TodF, 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase; XylE, catechol dioxygenase.
966 FEBS Journal 272 (2005) 966­974  2005 FEBS
less well studied. For example, there have been no systematic
substrate specificity studies of HPDA hydratases,
although HPDAs substituted with methyl or
chlorine groups can arise from the transformation of
certain aromatics such as p-xylene or polychlorinated
biphenyls (PCBs) [4]. In addition, the role of the essential
divalent metal ion in the hydratase has not been
resolved.
Mechanistic studies using XylJ, the HPDA hydratase
in the xylene degradation pathway of Pseudomonas putida
pWW0, show that the enzyme catalyzes deuterium
exchange of the C3 and C5 protons of the dienol substrate
[5,6]. The related MhpD (47% amino acid
sequence identity to XylJ) is strongly inhibited by
oxalate [7]. This led to the proposed mechanism
whereby the dienol substrate is first tautomerized to a
cis-keto anion intermediate, which then undergoes the
Michael addition of water. The hypothesis that the keto
intermediate has a cis rather than trans geometry is
based on the observation that the enzyme cannot
hydrate the trans-keto form of HPDA that is spontaneously
formed in aqueous solution [7,8]. The function of
the metal ion in the hydratase is not clear in these studies.
MhpD was not inhibited by crotonyl hydroxamate,
suggesting that substrate does not bind to the metal ion
in a bidentate manner i.e. through both the C1-carboxyl
and C2-carbonyl oxygens [7]. Based on mechanistic
consideration, interaction of the divalent cation with
the C2-carbonyl oxygen is expected to stabilize the
enolate anion transition state. Interaction with only the
C1-carboxyl, on the other hand, may aid in substrate
binding. The number of cations bound per enzyme active
site has not been determined in any HPDA hydratase
and it is therefore unclear if the metal ion(s) has
one or both of the proposed functions.
Here we report the overexpression and purification
of recombinant BphH (53 and 71% amino acid
sequence similarity to MhpD and XylJ, respectively), a
HPDA hydratase from the polychlorinated biphenyls
(PCBs) degradation pathway in Burkholderia xenoverans
LB400 [9,10]. The availability of large quantities of
monofunctional enzyme allows us to determine, for the
first time, the number of metal ions bound to each
hydratase by atomic absorption spectroscopy. We
measured the dissociation constants of Mg2+
and
Mn2+
and the influence of each metal cofactor on the
kinetic parameters for HPDA hydration. We also generate
and test the specificity of the enzyme towards
alternative substrates with chlorine and methyl functional
groups at the 5-position. Together, these results
provide insight regarding the mechanism, the substrate
binding site and the role of the metal cofactor in the
enzyme.
Results
Cloning, expression and purification of BphH
The bphH gene was inserted into the expression vectors
pT7-7 [11], pEMBL18 [12] and pVLT31 [13], which
contain the T7, lac and tac promoters, respectively.
The pT7-7 and pEMBL18 constructs were transformed
into Escherichia coli BL21 (kDE3) and E. coli DH5a,
respectively. Attempts to produce BphH in these
E. coli strains, at 37 °C and 18 °C, led to insoluble
inclusion bodies formation. BphH can, however, be
expressed in soluble form from Pseudomonas putida
KT2442 using the pVLT31 construct. The enzyme constitutes
about 20% of the soluble proteins in this bacterium
as estimated from SDS / PAGE. Overproduced
BphH was purified by anion exchange, hydrophobic
interaction and gel filtration chromatography to about
95% homogeneity with a yield of 38.8 mg of enzyme
per liter of cultured cells (Table 1, Fig. 2).
General properties of BphH
The subunit molecular mass of BphH, as determined
by SDS/ PAGE, is 27  1 kDa. This is in agreement
with the predicted molecular mass of 27.3 kDa, based
on its amino acid sequence. By gel filtration, the native
molecular mass of BphH is 221  25 kDa, suggesting
Table 1. Purification of BphH. BphH was purified from 1 L of cells.
Assays contain 0.2 mM HPDA and 3 mM MgSO4 in 100 mM potassium
phosphate buffer, pH 6.0. Substrate utilization was monitored
at 265 nm using the determined extinction coefficient of
15.85  0.29 mM
)1
cm)1
for HPDA. One unit is the enzyme
required to transform 1 lmol of HPDA substrate in 1 min.
Step
Protein
(mg)
Activity
(U)
Specific
activity
(Umg)1
)
Recovery
(%)
Purification
(n-fold)
Cell extract 508 4.16  104
81.9 100 1.00
SourceTM
15Q 81.7 2.44  104
306 59.0 3.74
PhenylSepharoseTM
44.9 2.37  104
528 57.0 6.45
SuperdexTM
200 38.8 2.07  104
533 49.7 6.51
Fig. 1. Reaction catalyzed by HPDA hydratase. Carbon numbering
of the HPDA substrate is indicated.
P. Wang and S. Y. K. Seah Characterization of BphH
FEBS Journal 272 (2005) 966­974  2005 FEBS 967
that the enzyme adopts an octameric quartenary structure,
assuming that it is globular.
The enzyme has no activity when divalent cations
are excluded from the enzyme assay mixture. Therefore
the native divalent cation, essential for the enzyme's
activity, is not tightly bound to the enzyme and is lost
during chromatographic purification of BphH. The
apoenzyme can, however, be reconstituted with Mg2+
in vitro and the specific activity of the purified enzyme
in the presence of saturating amounts of Mg2+
is
533 Umg)1
.
The relative specific activity of BphH was tested
with different divalent cations (Fig. 3). CuCl2 and
CuSO4 did not activate BphH activity. Activity of the
enzyme is highest with Mg2+
followed by Mn2+
. This
is in contrast to the homologous hydratase, MhpD,
where it is reported that the enzyme has the highest
activity with Mn2+
as cofactor [7]. Chloride or sulfate
salts of the respective divalent cations appear to have
no significant effect on the specific activity of the
BphH.
The activity of BphH-Mg2+
was determined at pH
values between 4.0 and 9.0, using constant ionic
strength pH buffers (Fig. 4). The enzyme has relatively
constant activity between pH 6.0 and 8.0, but activity
rapidly decreases below and above this pH range. In
contrast to the metal activation profile, the pH-rate
profile of BphH is similar to MhpD, suggesting that
both enzymes utilize similar residues with pKa values
of 6.0 and 8.0 for catalysis.
Number of metal ion bound per enzyme and
determination of coordination number
The equilibrium concentration of Mg2+
bound to
BphH was determined by atomic absorption spectroscopy.
A maximum of 1.12  0.09 mole of Mg2+
was
bound per mole of enzyme, suggesting that only a single
metal ion is present per enzyme subunit.
To determine the metal coordination number, UVvisible
spectroscopy was performed on the Co2+
BphH
complex. No strong absorbance features
between 500 nm to 700 nm was detected which is characteristic
of symmetry forbidden d­d transition and
indicative of an octahedral rather than a tetrahedral
metal coordination [14].
1 2 3 4
60
50
40
30
25
20
kDa
Fig. 2. Coomassie-blue stained SDS / PAGE of purified BphH. The
gel was loaded with samples of BphH from crude extract (lane 1);
preparation after anion exchange (lane 2); preparation after hydrophobic
column chromatography (lane 3), preparation after gel filtration
(lane 4). The molecular mass of the proteins in the standard
(lane 5) are indicated beside the gel.
Fig. 3. Relative activity of BphH with various divalent metal salts.
Activity obtained with MgCl2 is taken as 100%. Assays were performed
with 0.7 lgmL)1
BphH, 0.2 mM of HPDA and 1 mM of the
respective divalent metal ions in 100 mM phosphate buffer, pH 6.0.
Fig. 4. pH dependence of BphH activity. Assays were performed
as described in Experimental procedures. Spontaneous substrate
tautomerization rate is too rapid at pH above 9.0 to reliably measure
the enzyme catalyzed transformation.
Characterization of BphH P. Wang and S. Y. K. Seah
968 FEBS Journal 272 (2005) 966­974  2005 FEBS
Metal ions affinity and kinetic constants
for HPDA
Binding affinities of BphH towards Mg2+
and Mn2+
were determined by steady-state kinetic analysis of the
metal-dependent hydration of HPDA. The data can be
fitted to a single straight line in a Scatchard plot consistent
with the presence of only one metal ion in each
enzyme subunit (Fig. 5). From the Scatchard plot, the
dissociation constant of Mn2+
is determined to be
3.02  10)6
m, which is two orders of magnitudes
lower than for Mg2+
(Kd  5.74  10)4
m). Interestingly,
even though the enzyme's affinity for Mg2+
is
weak, BphH has the highest specific activity when saturated
with this metal ion.
The influence of the two metal ions on the kinetic
parameters for HPDA hydration by BphH is shown in
Table 2. The Km values were similar with either metal
cofactor. On the other hand, the kcat value is higher by
1.7-fold with Mg2+
. This suggests that the nature of
the metal ion has a predominant influence on catalysis
rather than substrate binding.
Substrate specificity of BphH
Substrate specificity of Mg2+
saturated BphH was
determined by steady state kinetic analysis. As HPDAs
are unavailable commercially, the substrates were
generated enzymatically from 4-methylcatechol and
4-chlorocatechol using the purified catechol dioxygenase,
XylE [15] and 2-hydroxy-6-oxohepta-2,4-dienoate
hydrolase, TodF [16].
The BphH activity data for 5-methyl-HPDA and
5-chloro-HPDA can be fitted to classical MichealisMenten
equation and the kinetic parameters are
summarized in Table 3. Specificity constants for
5-methyl-HPDA and 5-Cl-HPDA are lowered eightand
67-fold, respectively, in comparison to the unsubstituted
HPDA (Table 2). Interestingly, the lower
specificity of the enzyme towards the 5-substituted substrates
is mainly attributed to low turnover numbers
(kcat), as the Michealis constants (Km) are increased by
only about 1.3-fold. Thus, BphH active site can
Fig. 5. Dependence of BphH activity in varying concentrations of
(A) Mg2+
and (B) Mn2+
. Assays were performed with 100 mM
phosphate buffer, pH 6.0 containing 0.2 mM HPDA and
0.35 lgmL)1
BphH. The same data are represented as a Scatchard
plot (inset).
Table 2. Kinetic parameters for HPDA hydration catalyzed by BphH
saturated with Mg2+
or Mn2+
. Assays were performed at 25 °C
and they contained 3 mM divalent cation and 0.35 lgmL)1
BphH in
100 mM potassium phosphate buffer, pH 6.0.
Metal
cofactor
Km (lM)
HPDA kcat (s)1
)
kcat / Km
(M
)1
s)1
 10)6
)
MgSO4 31.7  1.3 361  6 11.4  0.6
MnSO4 38.6  1.4 215  4 5.6  0.3
Table 3. Specificity of BphH towards 5-substituted HPDA substrates.
Assays contained 3 mM MgSO4 in 100 mM potassium
phosphate buffer, pH 6.0. Substrate conversion was monitored at
the wavelength of maximum absorbance for each substrate, which
is 276 nm for 5-methyl-HPDA and 278 nm for 5-chloro-HPDA.
Extinction coefficients of 5-methyl-HPDA and 5-chloro-HPDA are
determined as described in Experimental procedures and found to
be 16.51  0.17 mM
)1
cm)1
and 13.57  0.69 mM
)1
cm)1
, respect-
ively.
Substrate Km (lM) kcat (s)1
)
kcat / Km
(M
)1
s)1
 10)6
)
5-Methyl-HPDA 40.8  2.6 60.5  1.6 1.5  0.1
5-Chloro-HPDA 45.9  4.5 7.7  0.3 0.17  0.02
P. Wang and S. Y. K. Seah Characterization of BphH
FEBS Journal 272 (2005) 966­974  2005 FEBS 969
accommodate the larger 5-substituted substrates. However,
the presence of electron withdrawing chlorine
substituent at the 5-position resulted in an eightfold
lower kcat value compared with the methyl substituted
substrate.
Discussion
HPDA hydratase, an enzyme in the meta-cleavage
pathway of aromatic compounds, catalyzes a hydration
reaction via a proposed anion transition state.
Previous studies with MhpD, suggest that this hydratase
may bind two divalent cations, one interacting
with the C2-carbonyl oxygen and the other to C1-carboxyl
oxygens [7]. Due to insufficient amounts of purified
enzyme, quantitative metal binding studies have
not been performed in MhpD. Another HPDA hydratase,
XylJ, on the other hand, can only be expressed as
a complex with a decarboxylase (XylI), the preceding
enzyme in the xylene degradation pathway [5]. The
binding of divalent cations by the decarboxylase precludes
metal binding studies of the hydratase in that
complex.
In this study, BphH, the hydratase in the PCBs
degradation pathway of B. xenoverans LB400, was
overexpressed and purified with high yield. The enzyme
is active without any associated enzymes. Using atomic
absorption spectroscopy, we show that the apoenzyme
binds to a single Mg2+
per enzyme subunit. Data from
a Scatchard analysis of the metal-dependent hydration
of HPDA by BphH can be fitted into a single straight
line, also indicating that there is only one metal ion
bound to the enzyme in the presence of substrate.
UV-visible spectra of the Co2+
­BphH complex demonstrate
that the coordination of the single metal ion in
the enzyme is octahedral rather than tetrahedral.
Divalent metal ions activate BphH in the following
order of effectiveness: Mg2+
, Mn2+
, Co2+
, Zn2+
and
Ca2+
. This differs from the metal activation profile
reported for MhpD, which is 1.6-fold more active with
Mn2+
than with Mg2+
[7]. In addition, the affinity of
MhpD for Mn2+
appears to be weak, requiring 10 mm
excess of Mn2+
to achieve maximum enzyme activity.
In comparison, the concentration of Mn2+
required
for maximal BphH activity is about 20 lm. Co2+
and
Zn2+
appear to activate MhpD to a similar extent,
whereas activity of BphH is threefold higher with
Co2+
than with Zn2+
. Despite this difference in metal
activation profile, the pH dependence of the enzyme
activity and the kinetic constants of HPDA hydration
for each enzyme, using the respective optimum metal
ion cofactor, are similar. For example, the Km value of
41 lm and kcat of 450 s)1
were reported for MhpD
with Mn2+
cofactor [7], which is of the same order of
magnitude as the corresponding values determined for
BphH with Mg2+
as cofactor. Sequence divergence
between the two enzymes (amino acid sequence identity
between BphH and MhpD is 37%) appears therefore
to affect mainly their respective metal cofactor
preferences. Despite the higher dissociation constant of
BphH for Mg2+
, this metal ion is the likely physiological
cofactor for the enzyme because it is highly abundant
in cells (10)3
m for Mg2+
compared with 10)8
m
for Mn2+
) [14].
In order to determine the role of the divalent metal
ion in the enzyme, we performed steady-state kinetic
analysis of HPDA hydration by BphH-Mg2+
and
BphH-Mn2+
. Mg2+
is smaller than Mn2+
by 0.11 A°
and it therefore has a higher charge density [17]. However,
the nature of the metal ion was observed to only
affect turnover number (kcat) and not the Km of the
HPDA substrate. Therefore, the metal ion has a catalytic
rather than just a substrate binding role. Possible
roles of the cation include the activation of water for
the hydration reaction and / or the stabilization of the
anion transition state. In the latter case, if the substrate
coordination to the metal ion is not bidentate,
interaction of the single metal ion in the active site
with the substrate C2-carbonyl oxygen instead of the
C1-carboxyl oxygens, will provide the optimal stabilization
of the proposed enolate anion transition state.
Therefore, the higher catalytic rate observed for Mg2+
could be due to its higher Lewis acidity compared with
Mn2+
. However, it should be noted that in MhpD,
activity with Mn2+
is higher than with Mg2+
, suggesting
that other factors, such as positions of the metal
ion due to differences in active site residues among
members of this group of hydratases, may also have a
bearing on catalytic rate.
Substrate specificity of the first four enzymes in the
meta-cleavage pathway has been extensively studied
[4,18­20]. In contrast, no comparative substrate specificity
data of the last three enzymes in the pathway are
available. B. xenoverans LB400 is a PCBs degrading
bacterium and it is therefore of interest to determine if
chlorinated HPDA is transformed by BphH. The Km
value for 5-chloro-HPDA is about 1.4-fold higher than
the unsubstituted substrate, while kcat value is lower
by 47-fold. Interestingly, the Michealis constant for
5-methyl-HPDA is also similar to 5-chloro-HPDA, but
the turnover number for this substrate is eightfold
higher than 5-chloro-HPDA. This result demonstrates
that the active site of BphH can accommodate the
larger substrates, but catalytic rate is reduced when the
substrate is substituted with an electron withdrawing
group. Possible explanations include a rate limiting
Characterization of BphH P. Wang and S. Y. K. Seah
970 FEBS Journal 272 (2005) 966­974  2005 FEBS
proton donation to the enolate intermediate, due to
inductive stabilization of the anion by the electron
withdrawing substituent. This is in line with previous
proposal of a step-wise hydration mechanism in the
HPDA hydratases [7].
Microorganisms that possess the meta-cleavage pathways
are useful for bioremediation of aromatic pollutants.
The availability of an expression system for
BphH reported in this work will enable future systematic
study of structure­function relationships through
site-directed mutagenesis. This may form the basis for
protein engineering efforts to improve the enzyme and
pathway for efficient degradation of a wide range of
aromatic pollutants.
Experimental procedures
Chemicals
Catechol, 4-methylcatechol and 4-chlorocatechol were
from Sigma-Aldrich (Oakville, Ontario, Canada). Restriction
enzymes, T4 DNA ligase and Pfx polymerase were
from Invitrogen (Burlington, Ontario, Canada) or New
England Biolabs (Pickering, Ontario, Canada). All other
chemicals were of analytical grade and were obtained from
Sigma-Aldrich and Fisher Scientific (Nepean, Ontario,
Canada).
Bacterial strains and plasmids
Strains used for DNA manipulation or protein expression
included E. coli DH5a [21], E. coli BL21(kDE3) [22] and
P. putida KT2442 [23]. Plasmids used in this work were
pT7-7 [11,22], pEMBL18 [12], pET28a (Novagen, EMD,
Biosciences Inc., San Diego, CA, USA) and pVLT31 [13].
E. coli was grown at 37 °C while P. putida was grown at
30 °C. Recombinant E. coli strains were propagated in
Luria­Bertani media supplemented with the appropriate
antibiotics at concentrations of 100 lgmL)1
for ampicillin,
15 lgmL)1
for tetracycline and 50 lgmL)1
for kanamycin.
Recombinant P. putida were grown on Luria­Bertani medium
supplemented with tetracycline (15 lgmL)1
) and rifampicin
(50 lgmL)1
).
DNA manipulation
DNA was purified, digested and ligated using standard protocols
[24]. N-Terminal His6-tagged TodF was constructed
by inserting the previously cloned gene [25] into the NdeI
and HindIII sites of the expression vector pET28a. The
bphH gene was amplified by PCR using the primers with
the respective sequences 5-CCCGCATATGACCCCTGA
ACTG and 5-CCCCAAGCTTCAGTGAAAGCGCAC.
NdeI and HindIII restriction sites are underlined. The PCR
reaction contain 10 ng of template pDD5301 [10], 0.8 units
of Pfx polymerase (Invitrogen), 20 nm amounts of each
dNTP and 100 pmol of each primer in a total volume of
100 lL. The following amplification profile was followed:
94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s,
50 °C for 30 s, and 68 °C for 1 min and finally 68 °C for
10 min. The amplified fragment was purified using the concert
rapid PCR purification system (Life Technologies, Inc.,
Burlington, Ontario, Canada), digested with NdeI and HindIII
and ligated to the same restriction sites in pT7-7. The
bphH gene from a positive clone was sequenced at the
Guelph Molecular Supercentre, University of Guelph, to
ensure no PCR-induced errors. The bphH gene was then
excised by XbaI and HindIII digestion and subcloned into
the vectors pEMBL18 and pVLT31.
Purification of BphH
Chromatography was performed on an A¨ KTA Explorer
100 (Amersham Pharmacia Biotech, Baie d'Urfe´ , Quebec,
Canada). Buffers containing 20 mm sodium Hepes, pH 7.5,
was used throughout the purification procedure unless indicated
otherwise.
The cell pellet (7 g) was resuspended in buffer and disrupted
by passing through French Press twice at an operating
pressure of 12 000 psi. The cell debris was removed by centrifugation
at 17500 g for 30 min. The clear supernatant was
filtered through a 0.45 lm filter and was loaded onto a
SourceTM
15Q (Amersham Pharmacia Biotech) anion
exchange column (2  13 cm), which has been equilibrated
with buffer containing 0.1 m NaCl. The column was washed
with a linear gradient of NaCl from 0.1 to 0.4 m over 10 column
volumes. Fractions containing BphH activity were eluted
at about 0.2 m NaCl. Active fractions were pooled and
concentrated by ultrafiltration with a YM10 filter (Millipore,
Nepean, Ontario, Canada) and was loaded onto Phenyl
SepharoseTM
hydrophobic interaction chromatography column
(1  18.5 cm), which was pre-equilibrated with 20 mm
sodium Hepes, pH 8.0 containing 0.1 m ammonium sulfate.
The column was washed first with a two-column volume of
the same buffer and then with a two-column volume linear
gradient of ammonium sulfate from 0.1 to 0 m followed by
five column volumes of ammonium sulfate-free buffer. Fractions
containing BphH activity were eluted at ammonium
sulfate-free buffer. Active fractions were pooled and concentrated
to about 4 mL by ultrafiltration with a YM10 filter
and then loaded onto HiLoadTM
26 / 60 SuperdexTM
200
prep gel filtration column (Amersham Pharmacia Biotech)
that had been pre-equilibrated with buffer containing 0.15 m
NaCl. The column was eluted with the same buffer at a flow
rate of 3 mLmin)1
. Fractions containing BphH activity were
pooled and concentrated as before. Purified BphH was
stored frozen in aliquots at )20 °C in 20 mm sodium Hepes
buffer, pH 7.5 and its activity remained stable for at least
three months.
P. Wang and S. Y. K. Seah Characterization of BphH
FEBS Journal 272 (2005) 966­974  2005 FEBS 971
Purification of catechol 2,3-dioxygenase (XylE)
Catechol 2,3-dioxygenase (XylE) was partially purified from
recombinant E. coli JM101 containing the plasmid pAW31
[15]. The harvested cells from 2 L culture were resuspend in
cell disruption buffer containing 20 mm sodium Hepes,
pH 7.5, 10% isopropanol, 2 mm dithiothreitol and passed
through French press twice. After centrifugation at
20 000 g for 30 min, the supernatant was filtered with a
0.45 lm filter. The initial NaCl concentration in the crude
extract was adjusted to 0.25 m and then loaded onto a
SourceTM
15Q (Amersham Pharmacia Biotech) anion
exchange column (2  13 cm), which has been equilibrated
with buffer containing 0.25 m NaCl. The column was
washed with a linear gradient of NaCl from 0.25 to 0.4 m
over 10 column volumes. Fractions containing XylE activity,
based on their ability to transform catechol to a yellow
product, were eluted at about 0.3 m NaCl, and were pooled
and concentrated by ultrafiltration with a YM10 filter.
Fresh iron(II) ammonium sulfate solution was added to
final concentration of 0.5 mm, and the enzyme was aliquoted
and stored at )20 °C.
Purification of hydrolase TodF
The recombinant N-terminal His6 tagged TodF was
expressed in E. coli BL21(kDE3) and purified by Ni­NTA
agarose resin according to the nondenaturing procedure
recommended by the supplier (Qiagen, Inc., Mississauga,
Ontario, Canada). His6-TodF was eluted with 50 mm
sodium phosphate buffer (pH 8.0) containing 300 mm NaCl
and 100 mm imidazole. TodF activity was detected based
on its ability to transform yellow colored 2-hydroxy-6-oxohexa-2,4-dienoate
to the colorless product, HPDA. Fractions
containing TodF were pooled, concentrated by
ultrafiltration and washed repeatedly with 20 mm sodium
Hepes, pH 7.5, to remove imidazole from the solution. The
concentrated enzyme (about 5 mgmL)1
) was stored frozen
in aliquots at )80 °C.
Preparation of HPDA and substituted HPDA and
determination of extinction coefficients
HPDA and substituted HPDA were generated enzymatically
from catechol, 4-methyl catechol and 4-chlorocatechol by
purified XylE and TodF, in 100 mm potassium phosphate
buffer, pH 6.0. Reaction was monitored by the increase
in UV absorbance. When maximum UV absorbance was
reached, the reaction was quenched by addition of concentrated
HCl to pH 1.0. HPDA was then extracted into ethyl
acetate and concentrated using negative pressure following
a previously described protocol [7,26]. The 2-hydroxy-6oxohexa-2,4-dienoate
(HODA) product from XylE cleavage
of 4-cholorocatechol is also purified by ethylacetate extraction
and analyzed by proton NMR and COSY using a Bruker
Avance 600 spectrometer (Milton, Ontario, Canada).
The resultant spectra confirmed that ring-cleavage occurs
between carbon 3 and 4 forming 5-chloroHODA. 1
H NMR
(acetone-d6): 9.54 (s, 1H), 7.89 (d, 1H, J  11.4Hz), 6.68
(d, 1H, J 11.4Hz) p.p.m.
Extinction coefficients of HPDA and substituted HPDA
were determined by adding XylE and TodF stepwise to
solutions containing known amounts of catechol and determining
the resulting UV absorbance. Complete cleavage of
catechol by XylE and the amounts of HODAs generated
were verified based on the extinction coefficients reported
previously [25].
Determination of protein concentration, purity
and molecular mass
Protein concentrations were determined by the Bradford
assay [27] using bovine serum albumin as standards.
SDS / PAGE was performed and stained with Coomassie
blue according to established procedures [28]. The Bench-
MarkTM
Protein Ladder (Invitrogen) containing proteins
ranging from 10 to 220 kDa was used as a molecular mass
marker. To determine molecular mass of BphH, gel filtration
was performed on a SuperdexTM
200 column (Amersham
Pharmacia) using 20 mm sodium Hepes buffer (pH 7.5) containing
0.15 m NaCl as equilibration and elution buffer. The
standard curve consists of the following proteins (Sigma):
cytochrome c (molecular mass  12.4 kDa), carbonic
anhydrase (molecular mass  29.0 kDa), bovine serum albumin
(molecular mass  66.0 kDa), alcohol dehydrogenase
(molecular mass  150.0 kDa), and b-amylase (molecular
mass  200.0 kDa).
Kinetic assays and substrate specificity
determination of BphH
BphH activity assay was performed by following the
HPDA substrate utilization as previously described [7]. All
kinetics assays were performed at least in duplicate at
25 °C using a Varian Cary 100 spectrophotometer equipped
with a thermojacketed cuvette holder. One unit of enzyme
represents the amount of protein that convert 1 lmol of
substrate to product in 1 min.
The standard activity assay during BphH purification
was performed in a total volume of 1.0 mL and contained
100 mm potassium phosphate buffer pH 6.0, 3 mm MgSO4
and 0.2 mm HPDA. Activity was followed spectrophotometrically
by the rate of decrease in absorbance at
265 nm. The background tautomerization rate of the substrate
determined without the addition of BphH was subtracted
from the former value to obtain the enzyme
catalyzed rate. Assays to determine the specificity of BphH
towards various HPDA substrates were carried out under
Characterization of BphH P. Wang and S. Y. K. Seah
972 FEBS Journal 272 (2005) 966­974  2005 FEBS
similar conditions except that the substrate concentrations
were varied from 0.1 to five times Km. Data were fitted to
a Michaelis­Menten equation by nonlinear regression
using the program Leonora [29]. The enzyme catalyzed
rate was at least fivefold higher than the spontaneous tautomerization
rates.
The pH dependence of BphH activity was performed
using the three component constant ionic strength buffer
containing 0.1 m Tris, 0.05 m acetic acid, and 0.05 m Mes
[30]. Activity of the enzyme was tested spectrophotometrically
as above over the pH range of 4.0­9.0, at 0.5 pH unit
increments. Assay mixtures contain 0.35 lgmL)1
BphH,
200 lm HPDA and 3 mm MgSO4 in a total volume of
1 mL. The extinction coefficient of HPDA, used to calculate
the specific activity, was found to be constant over the
pH range and is the same as the value obtained with
100 mm potassium phosphate buffer, pH 6.0.
Determination of metal ion cofactor specificity
Purified apoenzyme (70 lgmL)1
) was incubated on ice for
15 min with the respective divalent metal salts at 100 mm
concentrations: MgCl2, MgSO4, MnCl2, MnSO4, CoCl2,
CoSO4, ZnCl2, ZnSO4 and CaCl2. After incubation the
samples were assayed for activity in a final volume of 1 mL
containing 100 mm phosphate buffer, pH 6.0, 0.2 mm
HPDA and 1 mm of the respective metal ions.
The dissociation constants for Mg2+
and Mn2+
are
determined as follows. The enzyme was incubated and
assayed with the respective divalent metal salts at concentrations
varying from at least 0.5  Kd to 20  Kd. HPDA
concentration was fixed at 0.2 mm and amount of enzyme
in the assay is 0.35 lgmL)1
. All other conditions were the
same as standard activity assay. Dissociation constants (Kd)
were calculated based on the Scatchard plot (V / [M] vs. V)
from the equation
V=M  V=Kd  Vmax=Kd
where V is the initial velocity at various concentrations of
metal ions [M], and Vmax is the maximal catalytic rate at
saturating metal ion concentration [31].
Determination of number of magnesium ions
bound per enzyme subunit
Enzyme samples for analysis by atomic absorption spectroscopy
were prepared according to Petrovich et al. [32]. Buffer
and salt solutions are all prepared using metal-free
water (Fisher Scientific). Briefly, 0.4 mg of enzyme was
mixed with MgSO4 ranging from 0 to 3 mm in a total volume
of 400 lL of 20 mm sodium Hepes buffer, pH 7.5.
The free and bound metal ions were separated by ultrafiltration
to about 200 lL each with Amicon Microcon-30
concentrators. Saturation of Mg2+
in the enzyme was verified
by activity assays that demonstrate the attainment of
maximal HPDA transformation activity. Both the effluent
and the enzyme solutions were then diluted in 20 mm
sodium Hepes pH 7.5 and analyzed for Mg2+
content using
a PerkinElmer AAnalyst800 atomic absorption spectrophotometer
(Woodbridge, Ontario, Canada) operating in
oxy-acetylene flame mode with a 10 cm burner head. The
standard curve was generated using Mg2+
Atomic Absorption
Standard Solution (Fisher Scientific) prepared in the
same buffer as above.
Acknowledgements
This research was supported by a grant from the
National Science and Engineering Research Council of
Canada. S. Seah thanks the Canadian Foundation for
Innovation and Ontario Innovation Trust for infrastructure
support. We thank Valerie Robertson from
the University of Guelph NMR centre for NMR spectra
acquisition and Sean Langley for assistance with
the atomic absorption spectrometer.
References
1 Focht DD (1995) Strategies for the improvement of
aerobic metabolism of polychlorinated-biphenyls. Curr
Opin Biotechnol 6, 341­346.
2 Harayama S & Timmis KN (1990) Catabolism of aromatic
hydrocarbons by Pseudomonas. In Genetics of
Bacterial Diversity (Hopwood DA & Chater KF, eds),
pp. 151­174. Academic Press Inc., New York.
3 Ferrandez A, Garcia JL & Diaz E (1997) Genetic characterization
and expression in heterologous hosts of the
3-(3-hydroxyphenyl) propionate catabolic pathway of
Escherichia coli K-12. J Bacteriol 179, 2573­2581.
4 Seeger M, Zielinski M, Timmis KN & Hofer B (1999)
Regiospecificity of dioxygenation of di- to pentachlorobiphenyls
and their degradation to chlorobenzoates
by the bph-encoded catabolic pathway of Burkholderia
sp. strain LB400. Appl Environ Microbiol 65, 3614­
3621.
5 Lian HL & Whitman CP (1994) Stereochemical and isotopic
labeling studies of 4-oxalocrotonate decarboxylase
and vinylpyruvate hydratase ­ analysis and mechanistic
implications. J Am Chem Soc 116, 10403­10411.
6 Stanley TM, Johnson WH Jr, Burks EA, Whitman CP,
Hwang CC & Cook PF (2000) Expression and stereochemical
and isotope effect studies of active 4-oxalocrotonate
decarboxylase. Biochemistry 39, 718­726.
7 Pollard JR & Bugg TD (1998) Purification, characterisation
and reaction mechanism of monofunctional
2-hydroxypentadienoic acid hydratase from Escherichia
coli. Eur J Biochem 251, 98­106.
8 Harayama S, Rekik M, Ngai KL & Ornston LN (1989)
Physically associated enzymes produce and metabolize
P. Wang and S. Y. K. Seah Characterization of BphH
FEBS Journal 272 (2005) 966­974  2005 FEBS 973
2-hydroxy-2,4-dienoate, a chemically unstable intermediate
formed in catechol metabolism via meta cleavage in
Pseudomonas putida. J Bacteriol 171, 6251­6258.
9 Goris J, De Vos P, Caballero-Mellado J, Park J, Falsen
E Quensen JFIII,Tiedje JM & Vandamme P (2004)
Classification of the biphenyl- and polychlorinated
biphenyl-degrading strain LB400T and relatives as
Burkholderia xenovorans sp. nov. Int J Syst Evol Microbiol
54, 1677­1681.
10 Hofer B, Backhaus S & Timmis KN (1994) The
biphenyl / polychlorinated biphenyl-degradation locus
(bph) of Pseudomonas sp. LB400 encodes four additional
metabolic enzymes. Gene 144, 9­16.
11 Studier FW & Moffatt BA (1986) Use of bacteriophage
T7 RNA polymerase to direct selective high-level
expression of cloned genes. J Mol Biol 189, 113­130.
12 Dente L & Cortese R (1987) pEMBL: a new family of
single-stranded plasmids for sequencing DNA. Methods
Enzymol 155, 111­119.
13 de Lorenzo V, Eltis L, Kessler B & Timmis KN (1993)
Analysis of Pseudomonas gene products using
lacIq / Ptrp-lac plasmids and transposons that confer
conditional phenotypes. Gene 123, 17­24.
14 Cowan JA (1997) Inorganic Biochemistry: an Introduction,
2nd edn. John Wiley and Sons, Inc., New York.
15 Wasserfallen A (1989) Etude Biochemique et Genetique
de la specificite de al catechol 2,3-dioxygenase de Pseudomonas
putida. PhD Thesis. University of Geneva,
Geneva, Switzerland.
16 Menn FM, Zylstra GJ & Gibson DT (1991) Location
and sequence of the todF gene encoding 2-hydroxy-6oxohepta-2,4-dienoate
hydrolase in Pseudomonas putida
F1. Gene 104, 91­94.
17 Shannon RD (1976) Revised effective ionic radii and systematic
studies of interatomic distances in halides and
chalcogenics. Acta Crystallogr Section A 32, 751­767.
18 Barriault D, Vedadi M, Powlowski J & Sylvestre M
(1999) cis-2,3-Dihydro-2,3-dihydroxybiphenyl dehydrogenase
and cis-1, 2-dihydro-1,2-dihydroxynaphathalene
dehydrogenase catalyze dehydrogenation of the same
range of substrates. Biochem Biophys Res Commun 260,
181­187.
19 Seah SY, Labbe G, Nerdinger S, Johnson MR, Snieckus
V & Eltis LD (2000) Identification of a serine hydrolase
as a key determinant in the microbial degradation of
polychlorinated biphenyls. J Biol Chem 275, 15701­
15708.
20 Vaillancourt FH, Haro MA, Drouin NM, Karim Z,
Maaroufi H & Eltis LD (2003) Characterization of
extradiol dioxygenases from a polychlorinated biphenyldegrading
strain that possess higher specificities for
chlorinated metabolites. J Bacteriol 185, 1253­1260.
21 Hanahan D (1983) Studies on transformation of
Escherichia coli with plasmids. J Mol Biol 166, 557­580.
22 Tabor S & Richardson CC (1985) A bacteriophage T7
RNA polymerase / promoter system for controlled exclusive
expression of specific genes. Proc Natl Acad Sci
USA 82, 1074­1078.
23 Herrero ML, V & Timmis KN (1990) Transposon vectors
containing non-antibiotic resistance selection markers
for cloning and stable chromosomal insertion of
foreign genes in Gram-negative bacteria. J Bacteriol
172, 6557­6567.
24 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular
Cloning: A Laboratory Manual. Cold Spring Harbour
Laboratory Press, Cold Spring Harbour, New York.
25 Seah SY, Terracina G, Bolin JT, Riebel P, Snieckus V
& Eltis LD (1998) Purification and preliminary characterization
of a serine hydrolase involved in the microbial
degradation of polychlorinated biphenyls. J Biol Chem
273, 22943­22949.
26 Pollard JR, Henderson IMJ & Bugg TDH (1997) Chemical
and biochemical properties of 2-hydroxypentadienoic
acid, a homologue of enolpyruvic acid. Chem
Commun 19, 1885­1886.
27 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem
72, 248­254.
28 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680­685.
29 Cornish-Bowden A (1995) Analysis of Enzyme Kinetic
Data. Oxford University Press, New York.
30 Ellis KJ & Morrison JF (1982) Buffers of constant ionic
strength for studying pH-dependent processes. Methods
Enzymol 87, 405­426.
31 Newman JW, Morisseau C, Harris TR & Hammock
BD (2003) The soluble epoxide hydrolase encoded by
EPXH2 is a bifunctional enzyme with novel lipid phosphate
phosphatase activity. Proc Natl Acad Sci USA
100, 1558­1563.
32 Petrovich RM, Litwin S & Jaffe EK (1996) Bradyrhizobium
japonicum porphobilinogen synthase uses two
Mg(II) and monovalent cations. J Biol Chem 271, 8692­
8699.
Characterization of BphH P. Wang and S. Y. K. Seah
974 FEBS Journal 272 (2005) 966­974  2005 FEBS