 |
INTRODUCTION |
The esters of p-hydroxybenzoic acid, commonly named
parabens, are important preservative agents in the pharmaceutical,
cosmetic, and food industries. Parabens are active over a wide pH range (pH 4-8), are colorless, odorless, nonvolatile, stable, and have a low
acute and chronic toxicity and a broad spectrum of activity against
molds, yeasts, and bacteria (1). The antimicrobial activity of the
parabens increases with increasing alkyl chain length, although
limitations on the use of longer chain-length parabens are imposed by
their solubility in aqueous media. Hence, the methyl, ethyl, propyl,
and butyl parabens are more commonly used in commercial formulations.
Their anti-microbial effectiveness can be enhanced by combining two or
more parabens in a formulation, with the total paraben concentration
seldom exceeding 0.2-0.3% (2, 3).
Resistance to parabens can lead to the survival and proliferation of
microorganisms in commercial products that are normally well stabilized
with these antimicrobial agents. One mechanism of resistance of
bacteria toward parabens is hydrolysis of their ester bond. There are
many such reported cases of resistance to the parabens in the
literature. A strain of Cladosporium resinae isolated from a pharmaceutical suspension containing 0.2% of methyl paraben was able to hydrolyze this paraben (4). A strain of Pseudomonas aeruginosa was able to grow and degrade parabens
in an antimicrobial preparation used in the formulation of eye drops containing a mixture of methyl and propyl paraben at a total
concentration of 0.3% (5). The P. aeruginosa strain 396, isolated from an unpreserved oral formulation, was able to grow in the
presence of 0.1-0.2% of methyl and propyl parabens and to hydrolyze
propyl paraben to produce p-hydroxybenzoic acid (6). A
strain of Burkholderia cepacia isolated from an
oil-in-water emulsion containing 0.1-0.2% methyl and propyl parabens
was able to hydrolyze both parabens (7).
Although bacterial resistance to parabens has been reported in several
species, there are few reports of specific enzymes with the ability to
degrade parabens. The degradation of parabens by the resistant P. aeruginosa strain 396 was proposed to proceed through inducible
intra- and extracellular esterases, although these enzymes were not
isolated (6). An esterase purified from Aspergillus flavus
capable to hydrolyze depside ester linkages was shown to be
active against the methyl and ethyl parabens as part of its substrate
specificity profile, although microorganism resistance to the parabens
was not reported (8). Additionally, four different carboxyl esterases
capable of hydrolyzing parabens have been identified in human skin and
subcutaneous fat tissues. Two such esterases from subcutaneous fat
tissues were more active toward short chain parabens, whereas a third
one from transformed keratinocytes was more active toward longer
chain-length parabens such as butyl paraben (9).
A strain of Enterobacter cloacae was isolated from a
contaminated mineral supplement formulation containing 1700 ppm (11.2 mM) and 180 ppm (1.0 mM) methyl and propyl
paraben, respectively. The strain, named EM, was shown to be resistant
to parabens through the action of an esterase that hydrolyzed the
parabens to p-hydroxybenzoic acid (10). Here, we report the
purification and characterization of the PrbA esterase active against
parabens and other analogues.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All growth media, including tryptic soy broth and
tryptic soy agar were from Difco. The CM-Sepharose Fast Flow was
purchased from Amersham Biosciences, and the Avicell QMA and
SP-5W columns were from Waters (Milford, MA). The methyl, ethyl,
propyl, and butyl parabens and p-hydroxybenzoic acid as well
as 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK),1
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), diethyl
pyrocarbonate, and hydroxylamine were purchased from Sigma. All the
paraben analogues, p-nitrophenyl acetate,
p-nitrophenol, and n-propanol were purchased from
Aldrich. Methanol and ethanol were from EM Science (Gibbstown, NJ), and
n-butanol was from A&C American Chemicals (Ville
St.-Laurent, Quebec). Diisopropyl fluorophosphate (DFP) was obtained
from Calbiochem-Novabiochem. Sequencing-grade modified trypsin was
obtained from Promega (Madison, WI). Solid-phase extraction of tryptic
peptides was carried out on C4 or C18 Ziptips from Millipore (Bedford,
MA). Type D nanoflow probe tips for mass spectrometry were from
Micromass Canada (Pointe-Claire, QC).
Purification of PrbA--
A suspension of E. cloacae
strain EM was inoculated at an approximate cell density of 1 × 107 cells/ml in minimal Davis medium containing 1%
glucose. The cells were incubated overnight at 37 °C, subsequently
collected by centrifugation at 8000 rpm for 20 min, and resuspended in
20 mM MES-NaOH buffer, pH 5.5. The cells were lysed by a
3-fold French press treatment at a pressure of 1200 p.s.i.
followed by a centrifugation step at 18,000 rpm for 45 min and 2 ultracentrifugation steps at 40,000 rpm for 90 min each. The cell-free
protein suspension in 20 mM MES-NaOH buffer, pH 5.5, was
filtered through a 0.2-µm membrane (Millipore) before application on
the purification columns.
The first purification step used a CM-Sepharose Fast Flow column with a
60:40 ratio of 20 mM MES-NaOH, pH 5.5, and 20 mM MES-NaOH containing 1 M NaCl for the first
30 min and followed by a gradient that reached 100% NaCl after 60 min,
with a flow rate of 3 ml/min. Enzymatic activity was determined as
previously described (10), and the presence of PrbA was confirmed by
SDS-PAGE of the active fractions. The active fractions were pooled and
dialyzed into 20 mM Tris-HCl buffer, pH 8.4. The second
purification step, through an Avicell QMA column, used a gradient of
68:32 to 0:100 of 20 mM Tris-HCl, pH 8.4, and 20 mM Tris-HCl containing 1 M NaCl in 60 min at a
flow rate of 2 ml/min. The active fractions were pooled and dialyzed
into 20 mM MES-NaOH buffer, pH 5.5, and subsequently purified through a SP-5W column. The elution gradient was 100:0 to
30:70 of 20 mM MES-NaOH, pH 5.5, and 20 mM
MES-NaOH containing 1 M NaCl within 60 min at a flow rate
of 0.5 ml/min. The active fractions were subsequently pooled and stored
at
70 °C.
Substrate Specificity of PrbA--
All activity tests were done
in 5.0 ml of 0.05 M sodium phosphate buffer, pH 6.8, containing the selected paraben at concentrations of 0.5 or 1.0 mM and at 30 °C. The activity was measured after the
addition of 0.2 nM enzyme over an assay time of 5 min. The hydrolysis of the parabens and the amount of
p-hydroxybenzoic acid produced was quantified by HPLC
according to the method described previously (10). The specific
activities are expressed in mmol of p-hydroxybenzoic acid
produced/min/mg of protein. Specific activity measurements were done in
triplicate and are reported with the standard deviation. The
Km and Vmax values for the
parabens were determined using 10 sequential 2-fold dilutions of 1.0 mM solutions of methyl and propyl paraben and of a 0.5 mM solution of butyl paraben in 0.05 M sodium
phosphate buffer, pH 6.8. All Km and
Vmax values were calculated by nonlinear regression analysis with the software EZ-Fit 5.0 (Perella Scientific, Amherst, NH).
The Km and Vmax measurements
for p-nitrophenyl acetate were done in 2.0 ml of 7 consecutive 2-fold dilutions of a 1.0 mM solution in 0.05 M sodium phosphate buffer, pH 6.8, containing 4% acetone
to which 0.2 nM enzyme were added. The enzyme activity was
measured by monitoring the appearance of p-nitrophenol by absorbance readings at 405 nm. The p-nitrophenol produced
was quantified based on the calibration curve of a 1.0 mM
p-nitrophenol standard in sodium phosphate buffer containing
4% acetone. The background hydrolysis rate of p-nitrophenyl
acetate was subtracted from each assay.
All substrate analogues, including a reference solution of propyl
paraben, were prepared at stock concentrations of 100- 400 µM in 0.05 M sodium phosphate buffer, pH 6.8, and diluted to 50 µM. All assays with substrate analogues
were done with 0.2 nM enzyme as described above, with assay
times ranging from 5 to 15 min. The enzyme activity was measured as the
amount of substrate hydrolyzed as quantified by HPLC as described above.
Stability of PrbA--
The thermal stability profile of PrbA was
obtained by measuring the enzyme activity with 1.0 mM
propyl paraben according to the assay procedure above in 0.05 M sodium phosphate buffer at pH 6.8. The pH stability
profile was obtained by measuring the enzyme activity at 30 °C in
three different buffer systems. A 0.05 M sodium phosphate
buffer was used at pH 2.0-3.5 and at 6.0-7.5. A 0.05 M
sodium citrate buffer was used at pH 4.0-5.5, and a 0.05 M
sodium borate buffer was used at pH 8.0-9.5.
Transesterification Activity--
All transesterification assays
were done according to the same procedure as the substrate specificity
assays using 0.5 mM methyl or propyl paraben in 5.0 ml of
0.05 M sodium phosphate buffer, pH 6.8, containing either
0.5% of methanol, ethanol, n-propanol, or
n-butanol, or 5% of methanol or ethanol. All assays were
done in triplicate.
Inhibition of PrbA--
The measurement of enzyme activity after
inhibition with DFP, TPCK, TLCK, or diethyl pyrocarbonate was done
according to the same procedure as the substrate specificity assays,
using 100 µM propyl paraben. Each inhibitor, at a
concentration of 100 µM, was added at the mid-point of
the assay, or alternatively, the enzyme was preincubated with the
inhibitor for 10 min at 30 °C before the addition of the paraben.
Verification of the reversal of the diethyl pyrocarbonate-induced
inhibition by hydroxylamine was done by incubating the enzyme and
inhibitor for 10 min at 30 °C followed by another 10-min incubation
with 200 µM hydroxylamine and the subsequent addition of
the paraben. The inhibition constant Ki for TPCK and
TLCK was calculated from the relative Km values
observed for propyl paraben in the absence of the inhibitors and in the
presence of a fixed concentration of 100 µM TPCK or
TLCK.
The stoichiometry of the addition of DFP or TLCK to PrbA was verified
by incubating 5.6 µM PrbA in 0.05 M sodium
phosphate buffer, pH 7.0, with 100 µM DFP or 3 mM TLCK at 30 °C for 30 min. The modified enzyme was
subsequently purified from the sodium phosphate buffer and excess
inhibitor by solid-phase extraction with a C4 Ziptip using a 60:40
water:acetonitrile eluent with 0.1% acetic acid to which 10% acetic
acid was subsequently added. The molecular weight of the native and
modified enzymes was calculated from several mass spectrometry
measurements using a Quattro II Triple Quadrupole mass spectrometer
(Micromass Ltd., Manchester, UK) equipped with a Z spray interface in
nanospray. The molecular weight measurements were done in positive
electrospray ionization mode with a capillary voltage of 0.7-0.9 kV
and a cone voltage of 35-45 V.
Chemical Modification of Ser-189--
A concentration of 5.6 µM PrbA in 0.05 M sodium phosphate buffer, pH
7.0, was incubated with 100 µM DFP at 30 °C for 30 min. Subsequently, this mixture was added to 1 µg/µl trypsin in an equivalent volume of 0.025 M
NH4HCO3 buffer, pH 8.2, for an overnight incubation at 37 °C. The tryptic peptides were purified by
solid-phase extraction with a C18 Ziptip and eluted with a 60:40
water:acetonitrile mixture containing 0.1% acetic acid. Subsequently,
the concentration of acetic acid was increased to 10%.
Collision-induced dissociation studies of the tryptic peptides were
done with argon at 38-42-eV collision energy.
 |
RESULTS |
Purification of PrbA--
Three steps on a cell-free lysate from
E. cloacae strain EM were necessary to purify the esterase
PrbA to homogeneity (Table I). The first
step, through a CM-Sepharose column, resulted in a 7-fold increase in
the specific activity of the enzyme. The second step, using an Avicell
QMA column, was the most efficient in isolating PrbA, because the
specific activity after this step increased 55-fold relative to the
crude extract. The final step, with a SP-5W column, purified the enzyme
to homogeneity and slightly increased the specific activity, reaching
60-fold relative to the activity of the initial cell-free extract. The
enzyme was purified to homogeneity, as determined by SDS-PAGE, and its
molecular mass was calculated at 54.6 kDa. The molecular weight of the
enzyme was 54,619 ± 1 Da as measured by mass spectrometry.
Substrate Specificity of PrbA--
The specific activities of the
purified enzyme were determined with the most commonly used series of
parabens, from methyl to butyl paraben, and are shown in Table
II. The specific activity of PrbA is
greatest with ethyl paraben, reaching 2.71 mmol/min·mg. The specific
activity toward methyl paraben is nearly as high, reaching 91% that
with ethyl paraben. The activity declines with longer chain-length
parabens, reaching 70% activity with propyl paraben and 35% activity
with butyl paraben.
The Km and Vmax values were
determined for methyl paraben and for the longer chain-length propyl
and butyl parabens (Table II). The Km values
decrease ~2-fold as the alkyl chain length increases, from 0.88 mM with methyl paraben to 0.45 mM with butyl
paraben. The Vmax values decrease ~5-fold with
increasing chain length, from 0.15 mM/min with methyl
paraben to 0.031 mM/min with butyl paraben. The
Km value for p-nitrophenyl acetate is
~4-fold greater than that obtained with methyl paraben, whereas the
Vmax value for p-nitrophenyl acetate
is closest to the value obtained with butyl paraben.
The activity of PrbA toward several structural analogues of the
parabens was determined relative to the activity with propyl paraben
(Table III). The enzyme activity was
highest with propyl paraben, although the activity with methyl
3-hydroxybenzoate was nearly as high, reaching 97%. The second highest
activity was obtained with methyl 4-aminobenzoate, at 21%, and with
methyl vanillate, at 22%. Similar activities, reaching 12%, were
obtained with methyl 3,5-dihydroxybenzoate and ethyl
3,4-dihydroxybenzoate. The activity with the remaining substrates,
methyl benzoate, methyl 2-aminobenzoate, methyl 2-hydroxybenzoate, and
trans-methyl cinnamate ranged from 3 to 6%. PrbA was not
able to hydrolyze the ester bond of tert-butyl paraben nor
the amide bond of 4-hydroxybenzamide.
Stability of PrbA--
The activity profile of PrbA at different
temperatures shows an optimum activity at 31 °C and
90% activity
between the temperatures of 29 and 35 °C. The enzyme retained
80%
of its activity between 25 and 45 °C and more than 50% activity at
50 °C and 15 °C. The activity of PrbA at different pH values is
greatest at pH 7.0, and the enzyme maintains
90% of its activity in
the pH range 4.5-7.5. The enzyme retains ~70-80% of its activity
at pH values of 4.0 and 8.0. The activity decreases significantly
outside of this range, reaching 11% at pH 3.0 and 4% at pH 9.5. The
enzyme is completely inactivated at pH 2.5.
Transesterification Activity--
PrbA was able to transesterify
methyl or propyl parabens with 0.5% (v/v) of methanol, ethanol,
n-propanol, and n-butanol (Table IV). The rate of disappearance of propyl
paraben was 7% higher with methanol than in the buffer alone (1.73 and
1.61 mmol/min·mg, respectively), whereas the rate of formation of
p-hydroxybenzoic acid was 60% slower with methanol than
without (0.617 and 1.52 mmol/min·mg, respectively). The rate of
disappearance of methyl paraben (1.56 mmol/min·mg) was reduced by
23% in the presence of ethanol and by 60 and 77% with
n-propanol and n-butanol, respectively. The rate
of formation of p-hydroxybenzoic acid in the presence of
either of these three alcohols ranged from 17 to 43% that without any
alcohol (1.75 mmol/min·mg). The rate of formation of the
corresponding transesterified paraben was ~50% that of the rate of
disappearance of the original paraben with methanol, ethanol, or
n-propanol and reached only 14% with
n-butanol.
View this table:
[in this window]
[in a new window]
|
Table IV
Rates of transesterification of methyl paraben with 0.5% ethanol,
n-propanol, and n-butanol and of propyl paraben with 0.5% methanol
|
|
Additionally, PrbA was able to carry out the efficient
transesterification of propyl paraben with 5% (v/v) of methanol (Fig. 1). The yield of methyl paraben produced
after 2 h was 64% of the initial amount of propyl paraben in
solution (0.5 mM), whereas at the same time, the yield of
the side product p-hydroxybenzoic acid was minimized to 8%
of the amount of propyl paraben initially present. The yield of the
transesterified paraben was reproducible, as shown by the low margin of
error (Fig. 1). The same transesterification reaction with 5% ethanol
was much less rapid under the same conditions. After 2 h, only
13% of the initial propyl paraben was transformed, yielding 10% of
ethyl paraben and 2% of p-hydroxybenzoic acid relative to
the original amount of propyl paraben (results not shown).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Transesterification profile of 0.5 mM propyl paraben ( ) with 5% methanol, showing the
nearly complete disappearance of the paraben within 2 h and the
main product, the transesterified methyl paraben ( ), with a low
yield of the hydrolysis product p-hydroxybenzoic acid
( ). The error bars represent the S.D. of triplicate
measurements.
|
|
Inhibition of PrbA--
Total inhibition of PrbA activity occurred
when 100 µM DFP were added at the mid-point of a
hydrolysis assay with 100 µM propyl paraben. Complete
enzyme inhibition was also obtained under similar conditions by the
addition of 100 µM diethyl pyrocarbonate, and a 50%
restoration of activity was measured after the addition of 200 µM hydroxylamine to the diethyl pyrocarbonate-modified enzyme (results not shown). The addition of 100 µM TPCK
or TLCK under comparable conditions resulted in a 64 and 68% enzyme
inhibition, respectively. The inhibition constant Ki
for TPCK and TLCK was 292 and 202 µM, respectively. The
addition of TPCK or TLCK increased the Km value of
PrbA toward propyl paraben by 30-40% and had only a slight impact on
the Vmax value, suggesting that both of these
compounds act as competitive inhibitors of PrbA (results not shown).
The deconvoluted mass spectrum of PrbA after modification with DFP
showed only 1 peak at 54,784 ± 1 Da (results not shown). This
164-Da mass increase corresponds to the addition of 1 DFP molecule and
the loss of 19 Da from the fluoride leaving group. The single addition
of 164 Da confirmed a 1:1 stoichiometry of DFP addition to the enzyme.
The DFP-modified PrbA was totally inhibited.
The deconvoluted mass spectrum of TLCK-modified PrbA presented the peak
of the native enzyme at 54,619 ± 1 Da along with another at
54,948 ± 3 Da, with 17% intensity of the initial peak (results not shown). This mass increase corresponds within the experimental error to the addition of one TLCK molecule, with the loss of 35 Da from
the chloride leaving group. No other peak was observed corresponding to
multiple addition of TLCK. Only partial modification of PrbA could be
achieved at 3 mM TLCK because higher concentrations of TLCK
had a suppressing effect on the electrospray ionization of the enzyme.
This chemical modification resulted in a partial inhibition of enzyme
activity, reducing the activity by 51%.
Chemical Modification of Ser-189--
The addition of one DFP
molecule to PrbA made the identification of the modified serine
possible through collision-induced dissociation studies of the
tryptic peptide containing the active site motif GESAGG conserved
in many esterases. The full sequence of this tryptic peptide was
NIQSFGGDNHNVTLFGESAGGHSVLAQMASPGAK (amino acids 172-205)
with a molecular mass of 3399.7 Da. An ion corresponding to the [M + 4H]4+-charged state of this peptide was identified at
m/z 851, and a low intensity ion corresponding to
the [M + 3H]3+-charged state was found at
m/z 1134 (Fig.
2A). The ion at
m/z 851 was confirmed to represent the active
site peptide by collision-induced dissociation, where the
y61+-y111+, the
y132+-y242+, and the
y263+-y333+ ions were identified, spanning
nearly the entirety of the peptide sequence as follows:
IQSFGGDNXNVTLFGESAGGHXVLAQMA.
Additionally, the b202+-b212+ and the
b153+ - b173+ ions, corresponding to the
sequence FGEXXGG, were observed.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Mass spectrum of a tryptic digest of the
native (A) and DFP-modified (B)
PrbA. The ion at m/z 851 in the native
protein represents the +4 charged state of the peptide containing the
active serine. Upon DFP modification, an ion at
m/z 892 appears, which corresponds to the same
peptide with the addition of 164 Da, in the +4 charged state. The
additional ions from the native and DFP-modified peptide in the
+3-charged state are also observed.
|
|
The peak at m/z 851 present in the tryptic digest
of the native PrbA also represented two other peptides, which were
identified by collision-induced dissociation. These peptides did not
contain the serine active site motif. The first peptide, in a [M + 2H]2+-charged state, contained the sequence LSILMVDYWTNFAK
(amino acids 440-453) and had a molecular mass of 1699.9 Da. This
peptide was identified from the series of
y5+-y13+ and b5+-b9+
fragment ions. The second peptide, in a [M + 3H]3+-charged state, contained the sequence
KLPVMVWIPGGGLSSGSGNEYDASK (amino acids 97-121) and
had a molecular mass of 2549.9 Da. This peptide was identified from the
series of y5+-y17+ and
b5+-b9+ fragmentation ions.
Upon modification by DFP, an ion at m/z 892 appeared that was not present in the tryptic digest of the native
enzyme (Fig. 2B). This ion corresponds to the molecular mass
of the active site peptide with the addition of 164 Da from the DFP
molecule, corresponding to 3563.7 Da in the [M + 4H]4+-charged state. The [M + 3H]3+ ion, at
m/z 1189, was also observed at a low intensity.
The charged state of the m/z 892 ion was +4, as
confirmed by the 0.25 Da separation of its isotopic peaks. The
remaining ion at m/z 851 in the DFP-modified tryptic digest (Fig. 2B) is due to the presence of the two
unrelated peptides described above.
The tryptic digests of the native and DFP-modified PrbA permitted the
identification of a considerable number of peptides besides the active
site peptide. A total of 24 peptides were identified from the native
PrbA of a theoretical total of 42 tryptic peptides. An additional 5 peptides were identified from tryptic fragments that contained one
missed cleavage site, covering 70% of the entire enzyme. In the
DFP-modified PrbA, the total number of identified peptides was 23, with
an additional 11 peptides identified from fragments with one missed
cleavage site, covering 81% of the enzyme. Generally, the same
peptides were identified from the native and DFP-modified enzymes,
although their relative intensities were different (Fig. 2).
Collision-induced dissociation of the m/z 892 ion
from the DFP-modified PrbA confirmed that it contained an active serine residue involved in the catalytic site. Although the entire peptide contained four serine residues that DFP could possibly modify, the
serine of interest in the GESAGG motif was Ser-189. In the collision-induced dissociation profile of this peptide, fragmentation at Ser-189 corresponds to the y17 ion or b18 ion (Fig.
3A). The collision-induced
dissociation spectrum of m/z 892 showed the y122+-y242+ ions corresponding to the sequence
NVTLFGESAGGH, with a mass addition of 164 Da from the
y172+ (Ser) to the y122+ (His) ions, confirming
the addition of 164 Da to Ser-189 at m/z 866 (Fig. 3B). The y202+ ion
(m/z 1038) is not indicated, because its signal
is masked by the intense b182+ ion at
m/z 1040. As well, the
b112+-b182+ ions corresponding to the sequence
VTLF(G)ES were identified as well as the
b232+ and b242+ ions, which had an added mass
of 164. The missing b162+ ion and several other ions
between b182+ and b232+ correspond to the loss
of glycine residues, which are often difficult to detect. In addition,
the series of y51+-y171+ ions corresponding to
the sequence SAGGHSVLAQMA were identified, with a low
intensity signal at m/z 1733 for the
y171+ ion (Ser-189 + 164 Da) (results not shown). The shift
in mass at Ser-189 by 164 Da identifies the site of DFP modification, and the concurrent total loss of activity confirms that Ser-189 is the
active serine in the catalytic site.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Collision-induced dissociation of the active
site peptide from the DFP-modified PrbA. A,
fragmentation profile of the entire peptide, showing the series of
b-ions generated from the N terminus and the y-ions from the C
terminus. B, collision-induced dissociation spectrum showing
the series of y2+ and b2+ ions characteristic
of the active site peptide with the addition of 164 Da to Ser-189,
which corresponds to the y17 and b18 ion.
|
|
 |
DISCUSSION |
The esterase PrbA was purified from the paraben-resistant strain
of E. cloacae EM. The molecular mass of the purified enzyme, as measured by SDS-PAGE, was 55 kDa. This measurement was confirmed by
the theoretical molecular mass of the enzyme, calculated from the
sequence of amino acids deposited under the GenBankTM
accession number AAL82802, as 54,597 Da (11). The molecular mass of the
enzyme as measured by mass spectrometry was 54,619 ± 1 Da, which
corresponds to the addition of 22-23 mass units, possibly from a
sodium ion originating from the sodium phosphate buffer. The esterase
showed the highest specific activities toward the ethyl, methyl, and
propyl parabens, indicating a preference for shorter chain esters
(Table II). Although the decreasing Km values with
increasing alkyl chain length indicate a higher affinity for the longer
chain-length parabens, the higher Vmax values
with methyl paraben indicate that the rate of reaction is greatest with
shorter chain-length parabens. The affinity of the enzyme for
p-nitrophenyl acetate is relatively low, as indicated by a Km value 4-fold higher than that for methyl paraben
as well as by a low Vmax value.
PrbA showed an almost equal activity toward methyl 3-hydroxybenzoate as
for propyl paraben (Table III). However, the activity with methyl
2-hydroxybenzoate was only 3% that with propyl paraben, indicating
that the hydroxyl group must be placed in the meta or
para position but not in the ortho position for
efficient hydrolysis by PrbA. The presence of two hydroxyl or methoxy
group substituents in the para and/or meta
position still results in 12-22% residual activity. The absence of
any polar substituent at the para or meta
position leads to only 6% residual activity, as shown with methyl
benzoate. The hydroxyl groups at the para position can be
substituted by another electron-donating group such as an amine function, as with methyl 4-aminobenzoate, and PrbA still retained 21%
of its activity. The analogues methyl 2-hydroxybenzoate and methyl
2-aminobenzoate showed a much reduced reactivity, confirming that the
presence of a substituent at the ortho position results in
significant loss of enzymatic activity. Interestingly, PrbA was capable
of hydrolyzing methyl cinnamate, although in low yields, showing that
its activity was not limited only to benzoic ester analogues, as also
shown by its activity against para-nitrophenyl acetate. PrbA
could not cleave the ester bond of tert-butyl paraben due to
the steric hindrance at the carbonyl caused by the bulky t-butyl group, but utilization of this paraben is hampered
by its very limited solubility in water. The activity of PrbA seems to
be limited to ester bonds, because it could not cleave the amide bond
of the structurally related 4-hydroxybenzamide.
It has been previously shown that a P. cepacia strain that
could hydrolyze low concentrations of parabens (100 mg/liter) in 3 weeks could also perform the transesterification of ethyl paraben with
various alcohols (12). This transesterification was attributed to an
esterase, probably the same that hydrolyzed the paraben. However, the
enzyme responsible was not isolated, and the transesterification process, although efficient, required 3 days for 80% conversion of
ethyl paraben to methyl paraben with 0.4% of methanol (12). The
ability of PrbA to carry out paraben transesterification was then
investigated with a series of alcohols of increasing chain length
ranging from methanol to n-butanol. The efficiency and yield
of transesterification was greatest with methanol and ethanol (Table
IV) and decreased approximately by half with n-propanol. The
yield of butyl paraben obtained upon transesterification with n-butanol was the lowest, reaching only 6% of the yield
obtained with methanol. Furthermore, PrbA can carry out efficient
transesterification with methanol concentrations as high as 5% within
a short period of time (Fig. 1), achieving 64% transesterification in
2 h.
The residues involved in the active site of PrbA were investigated. The
catalytic site of many esterases contains a catalytic triad composed of
an active serine, histidine, and aspartic acid. The presence in the
catalytic site of the serine and histidine residues was investigated
using reagents known to react selectively with these active residues.
The protease inhibitor DFP is known to react selectively with active
serine residues (13). Modification of PrbA with DFP resulted in a
complete irreversible inhibition with a 1:1 stoichiometry as observed
by mass spectrometry. Analysis of the tryptic peptides of the
DFP-modified PrbA showed that the modified residue was Ser-189. This
serine is part of the sequence GESAGG, which corresponds to the
conserved GXSXG motif commonly found in carboxyl
esterases (14), further confirming the identity of this serine as part
of the active site of PrbA. An alignment of the 100 esterases most
closely related to PrbA with the protein-protein BLAST engine showed
that this motif was conserved among all these esterases.
The protease inhibitor TLCK reacts selectively with active histidine
residues (15). Only one molecule of TLCK was found in the modified
enzyme. This suggests that the histidine residue that reacted was part
of a catalytic triad. At the TLCK concentration used only 17% of the
enzyme was modified, as observed by mass spectrometry, whereas the
activity was reduced by 51%. This difference could be attributed to
the relative instability of TLCK-derivatized histidine, especially at
the low pH required (10% acetic acid) for efficient ionization in
electrospray. The protein-protein BLAST alignment of the 100 esterases
more closely related to PrbA showed that one residue, His-412, was
conserved in 98 of the 100 proteins. His-412 was also aligned with the
regions of the other esterases containing the motif
GDHXD. This motif is commonly found for the
histidine in the catalytic triad of serine hydrolases (14), although it
was not present within the sequence of PrbA itself.
A motif GXXXXEXG, often indicative of the
glutamate in the catalytic triad of serine hydrolases, was also located
in the same BLAST alignment described above. This region included the
highly conserved Glu-307 in PrbA, which was conserved in 98 of the 100 esterases. The glutamate 307 in PrbA was part of a slightly modified GXXXXEG motif, which was also found in several other
homologous esterases instead of the GXXXXEXG motif.
A small number of esterases have previously been shown to have the
ability to hydrolyze parabens, namely from P. cepacia, P. aeruginosa, or from human skin and subcutaneous fat
tissue as well as an esterase hydrolyzing depsi-peptide linkages from A. flavus (6, 7, 8, 9). However, none of these enzymes have
been isolated or characterized in their purified form, and hence, their
amino acid sequence and other biophysical parameters are unknown. The
esterase PrbA represents the first fully characterized carboxyl
esterase responsible for the hydrolysis of parabens, and it was shown
that the E. cloacae strain EM containing the prbA
gene was more resistant toward parabens than a E. cloacae reference strain that did not have a paraben-hydrolyzing activity (11).
PrbA is most active at physiological pH and at a temperature near
30 °C, which are conditions that often apply to commercial preparations containing parabens. Two prbA genes closely
homologous to the prbA gene found in E. cloacae strain EM have been identified in two
Enterobacter gergoviae strains recently isolated
in France (11). Both strains hydrolyzed parabens, and one of them was more paraben-resistant than a E. gergoviae reference strain.
This geographical separation might indicate that homologues of the prbA gene are more widespread than currently known. Hence,
the characterization of PrbA is an important step in understanding and
limiting the growth of paraben-resistant microorganisms in commercial formulations.