Purification and Characterization of PrbA, a New Esterase from Enterobacter cloacae Hydrolyzing the Esters of 4-Hydroxybenzoic Acid (Parabens)*

Nelly Valkova, François LépineDagger, Louisette Labrie, Maryse Dupont, and Réjean Beaudet

From the Institut Armand-Frappier, Institut National de la Recherche Scientifique, Université du Québec, Laval, Québec H7V 1B7, Canada

Received for publication, December 30, 2002, and in revised form, January 23, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 ± 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with Km and Vmax values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The Km and Vmax values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 °C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mM propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7- amino-2-heptanone (TLCK), with Ki values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


                              
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Table I
Specific activity of PrbA after each purification step

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.


                              
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Table II
PrbA activities toward the methyl to butyl parabens and p-nitrophenyl acetate

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.


                              
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Table III
Relative activity of PrbA toward paraben structural analogues

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.


                              
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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).


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Fig. 1.   Transesterification profile of 0.5 mM propyl paraben (open circle ) with 5% methanol, showing the nearly complete disappearance of the paraben within 2 h and the main product, the transesterified methyl paraben (diamond ), with a low yield of the hydrolysis product p-hydroxybenzoic acid (black-triangle). 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.


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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.


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* This work was funded in part by postgraduate fellowships from the Natural Sciences and Engineering Research Council and by the Fonds de la Recherche en Santé du Québec-Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.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.

Dagger To whom correspondence should be addressed: Institut Armand-Frappier, INRS, 531 Boulevard des Prairies, Laval, Québec H7V 1B7, Canada. Tel.: 450-687-5010; Fax: 450-686-5501; E-mail: francois_lepine@inrs-iaf.uquebec.ca.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M213281200

    ABBREVIATIONS

The abbreviations used are: TPCK, 1-chloro-3-tosylamido-4-phenyl-2-butanone; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; DFP, diisopropyl fluorophosphate; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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