Ligninase-Mediated Removal of Natural and Synthetic Estrogens from

Dec 12, 2008 - State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, People's R...
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Environ. Sci. Technol. 2009, 43, 374–379

Ligninase-Mediated Removal of Natural and Synthetic Estrogens from Water: I. Reaction Behaviors L I A N G M A O , †,‡ Q I N G G U O H U A N G , * ,‡ JUNHE LU,‡ AND SHIXIANG GAO† State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, People’s Republic of China and College of Agricultural and Environmental Sciences, Department of Crop and Soil Sciences, University of Georgia, Griffin, Georgia 30223, United States of America

Received June 27, 2008. Revised manuscript received October 14, 2008. Accepted November 3, 2008.

Our experiments revealed that a few natural and synthetic estrogens can be effectively transformed through reactions that are mediated by lignin peroxidase (LiP), an extracellular enzyme that is produced by a white rot fungus Phanerochaete chrysosporium and is widely present in the natural environment. We systematically assessed the reaction efficiencies at varying important conditions and identified the reaction products using mass spectrometry. In particular, we compared the reaction behaviors for systems variously containing natural organic matter and/or veratryl alcohol, a secondary metabolite that P. chrysosporium produces along with LiP in nature to play a role in mediating LiP activity. On the basis of the observed reaction behaviors and the molecular characteristics of the substrates and the enzyme, we postulate that the active binding site for estrogens is located within the LiP heme cavity, whereas that for veratryl alcohol is on the enzyme surface. Our study suggests that the processes mediated by LiP and other naturally occurring enzymes of similar nature may influence the environmental transformation and fate of estrogen contaminants. The findings in this study provide useful information for understanding LiP-mediated estrogen reactions and for potential development of novel enzymatic method to control estrogen contamination.

Introduction As an “emerging” class of contaminants, endocrine disrupting chemicals (EDCs) are attracting increased public concern and scientific interest due to their potential for impacting ecosystems and human health by disrupting reproduction, growth, and development (1-4). Natural estrogens, estrone (E1), 17β-estradiol (E2), and estriol (E3) and the synthetic estrogen, 17R-ethinylestradiol (EE2), are among the most potent endocrine disruptors and comprise a major source of potential EDC contamination (3-5). A nationwide survey (6) revealed a widespread occurrence of endocrine disruptors in natural water bodies and found estradiol and estrone concentrations as high as 200 and 112 ng/L, respectively. * Corresponding author phone:(770) 229-3302; fax:(770) 412-4734; e-mail: [email protected]. † State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University. ‡ College of Agricultural and Environmental Sciences, Department of Crop and Soil Sciences, University of Georgia. 374

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The characteristics that enable hormones to be physiologically functional, such as being relatively bioaccumulative, persistent, and capable of eliciting physiological responses at extremely low concentrations (∼ng/L) (1), make this group of chemicals a unique challenge from the perspective of water/wastewater treatment. Conventional treatment technologies are either futile or require very high inputs of energy and reagents to bring the trace contaminants from very low levels at which they usually occur to even lower safety levels (5, 7-10). Recent studies indicate that enzymatic reactions may provide promising alternatives to address EDCs and other emerging micropollutants (5, 7-11). Enzyme catalysis is generally highly efficient and specific, thus offering the possibility of chemically or biochemically transforming trace contaminants with reasonable consumption of reagents/cofactors and energy. Removal of various EDCs from water via enzymatic reactions catalyzed by horseradish peroxidase and laccase has been documented in recent publications (1, 5, 7-10). Lignin peroxidases (LiPs), also known as ligninases, comprise a class of enzymes that have been widely used in pollution control (12, 13). These enzymes play a unique role in the geochemical cycling of carbon by mediating lignin depolymerization, which is one of the only several pathways existing in nature to degrade lignin (13, 14). Certain lignindegrading fungi, such as white rot fungus Phanerochaete chrysosporium, produce these enzymes in nature, particularly when nutrient limitation conditions are present (12, 13). LiPs have strong oxidizing ability and are capable of catalyzing one- and two-electron oxidations of a wide range of chemicals, including those that exhibit strong reduction potentials such as lignin, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and aromatic dyes (12, 13, 15-17). As such, LiP-based biological treatment processes have been developed to remove lignin from wood pulp in the paper industry and proposed for the removal of various persistent organic pollutants in pollution control (12, 18). The reaction behaviors of several estrogens in ligninase-mediated processes were thoroughly investigated in this study. LiP contains an iron-porphyrin catalytic center, and followstheclassicalperoxidasecatalysismechanism(14,19-21), where the ferric resting enzyme is oxidized by two electrons of H2O2 to so-called Compound I (LiPI). Compound I contains ferryl iron and a porphyrin π cation radical, which can oxidize one substrate molecule by one electron with a concomitant reduction of the porphyrin π cation radical yielding Compound II (LiPII). Compound II can also oxidize one substrate by one electron and then return the enzyme to the resting state (a schematic depiction of the LiP catalytic cycle is presented in Supporting Information I). Both molecular dynamic simulations (22) and crystallographic refinement (14) indicate the existence of a heme cavity in LiP that can serve as the binding site for substrates. The presence of a substrate binding site in a heme cavity is also well established for plant peroxidases such as horseradish peroxidase, Coprinus cinereus peroxidase, and ascorbate peroxidase (23). However, the three-dimensional X-ray diffraction structure of fungal LiP shows that the heme is buried inside the protein with only a small opening connecting the active site with outside of the protein and this site may only accommodate small substrates (20, 24). This has promoted the suggestion that the classical heme-edge site may not be the only substrate interaction site present in LiP (23, 24) due to its ability to degrade many large molecules such as lignin. In this study, the possible binding sites in LiP for the estrogens under investigation were explored based on their catalytic reaction 10.1021/es801791v CCC: $40.75

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behaviors and the molecular characteristics of the substrates and the enzyme. It is a well-known and intriguing fact that veratryl alcohol (VA), a secondary metabolite that Phanerochaete chrysosporium produces along with LiP in nature, is itself a substrate of LiP and can mediate and/or stimulate the LiP-mediated oxidation of a number of substrates such as lignin, proteins, and a variety of aromatic compounds (16, 19, 20). Although VA is not large in size, abundant data prove that the binding site of VA is not in the heme cavity, but located on the LiP surface at or near Trp 171 (16, 20, 21, 23-25), and there exists a mechanism enabling long-range electron transfer from Trp171 to the heme (23-26). In this study, we experimentally examined how the presence of VA influenced the LiPmediated estrogen reactions. In addition, we further explored how LiP-mediated estrogen reactions may be influenced by natural organic matter (NOM) that is ubiquitously present in water. Our results indicated that the reaction systems respectively having VA present and absent were influenced by NOM in a different manner. We discussed this with regard to the possible enzyme binding modes for VA, NOM, and estrogen chemicals.

Experimental Section Materials. Steroid hormones E1 (CAS 53-16-7), E2 (CAS 50-28-2), E3 (CAS 50-27-1) and EE2 (CAS 57-63-6), veratryl alcohol (3, 4-Dimethoxybenzyl alcohol, 96%, CAS 93-03-8), and hydrogen peroxide (30 wt %, CAS 7722-84-1) were purchased from Sigma-Aldrich (St. Louis, MO). Molecular structures of E1, E2, E3, and EE2 are provided in Supporting Information II. An enzyme solution containing lignin peroxidase (EC 1.11.1.14) was produced by culturing Phanerochaete chrysosporium as described below. This enzyme solution was used in the experiments to test the removal efficiency of E2 under varying conditions. Purified LiP purchased from Sigma-Aldrich was used for all of the remainder experiments, which were intended to characterize the enzymatic reaction products and behaviors as described below. Acetonitrile (ACN) and methanol were HPLC grade solvents obtained from Fisher Scientific (Pittsburgh, PA). Citric acid (CAS 77-92-9) and dibasic sodium phosphate (CAS 7558-79-4) were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ). Suwannee River Fulvic Acid (SRFA) was obtained from the International Humic Substances Society (IHSS). USDA Center for Forest Mycology Research Program Northern Research Station provided P. chrysosporium BKM-F-1767 strain. Enzyme Production by Fungal Culture. P. chrysosporium was incubated in an N-limited culture under agitation at 37 °C following the same conditions as described by Tien et al. (13). The cultures were aerated with oxygen for 30 min each day during incubation. When LiP activity reached maximum on the sixth day of incubation, the culture was centrifuged at 10 000 ×g for 8 min at 4 °C, and the supernatant enzyme solution was collected and passed through 0.45 µm sterile filters. The enzyme solution was further concentrated by ultrafiltration using a Minimate Tangential Flow Filtration system (Pall Corporation, East Hills, NY) equipped with a 10-KDa cutoff membrane capsule. Enzyme Activity Assay. A colorimetric assay was used to quantify the activity of the LiP enzyme (13), in which veratryl alcohol was oxidized by LiP catalysis to veratraldehyde, the strong absorbance of which was measured for quantification (Beckman Du 640-B spectrophotometer, Beckman Instruments, Inc.) at 310 nm (molar extinction coefficient ) 9300 M-1 cm-1). The enzyme activity assay medium was made up of 400-µL tartarate buffer (pH 3.0), 400-µL LiP solution, 200µL 2-mM H2O2 and 200-µL 10-mM veratryl alcohol, and absorbance was recorded every 15 s for about 2 min. One

enzyme unit corresponds to the amount of LiP that oxidizes 1-µmol veratryl alcohol per minute. The assay protocol described above was also modified and used to explore the influence of NOM on LiP-mediated VA reaction. In these tests, SRFA was added to the assay medium specified above at 0, 1, 5, or 10 mg/L (TOC level) as model NOM, and the absorbance at 310 nm was recorded every 15 s for 7 min to investigate how VA conversion was influenced. Assessment of E2 Removal at Varying Reaction Conditions. Experiments were performed to assess E2 removal through reactions mediated by LiP present in the fungal culture extracts described above under different pH and H2O2concentration conditions. The reactions were carried out in glass test tubes as batch reactors under room temperature. Each reactor contained a 2-mL reaction medium prepared in 10-mM citrate-phosphate buffer solution (CPBS) that was adjusted to a prescribed pH. The reaction solution initially contained 10-µM E2, H2O2 at different concentrations, and the fungal culture extract to provide LiP activity. Hydrogen peroxide was added to each reactor as the last component to initiate the reaction, following which, the reactor was hand shaken for about 30 s and allowed to react for about 30 min prior to the addition of 2-mL methanol to terminate the reaction. The mixture of reaction medium and methanol was sampled for HPLC analysis described in Supporting Information III. Three replicate experiments were performed for each reaction condition. Experiments were also carried out using the same reactor setup and procedure described above to examine how the presence of VA and/or natural organic matter may influence LiP-mediated E2 removal. Purified LiP obtained from SigmaAldrich was used in these experiments, and the 2-mL reaction media were prepared in CPBS (pH 4.6; 10 mM) initially containing 0.01-mM H2O2, 0.0065-U/ml LiP, and 19-µM E2. The reactors also contained 1-mM VA and/or IHSS SRFA (5 mg/L TOC) as a model NOM for comparison of reaction effects in the absence or presence of these components. A series of reactors was prepared for each reaction condition tested, and triplicate reactors were scarified and sampled at 0.5, 3, 5, 10, 20, 30, 60, and 90 min. A 2-mL portion of methanol was added to a reactor to terminate the reaction at a sampling time, and the mixture of methanol and reaction solution was sampled for HPLC analysis to determine the E2 concentration remaining in the reactor. Reactors that had LiP absent served as blank controls. Product Identification. Samples used for product identification were prepared by reactions performed in a 25-mL flask reactor containing 10-mL of reaction solution prepared in CPBS (pH 4.6; 10 mM). The reactions were initiated with 19-µM E2, 10-µM H2O2, and varying dosages of LiP, and were allowed for 60 min. Purified LiP obtained from Sigma-Aldrich was used in these experiments. The product mixtures were then concentrated by solid phase extraction (SPE) and the extracts were characterized by a Waters Micromass Quattro Mass Spectrometer, with details given in Supporting Information III. Blank samples that did not contain LiP or E2 were also analyzed. Assessment of Michaelis-Menten Reaction Kinetics. Purified LiP obtained from Sigma-Aldrich was used in experiments to assess the initial reaction rates of each studied substrate at various initial concentrations, and the data were used to construct Michaelis-Menten curves and for associated kinetic analysis. Reactions were conducted under room temperature using glass test tubes as the reactors and following a scheme that we have used in our earlier studies (1), which is described in detail in Supporting Information III. Determination of Pertinent Molecular Descriptors. Molecular descriptors characterizing the physicochemical VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Measured KM and kCAT and Calculated Molecular Descriptors of the Substrates compound KM (µM) kCAT (s-1) volume (Å3) EHOMO ELUMO log P VA E1 E2 E3 EE2 Heme Cavity a

102.9a 2.559 0.898 4.925 14.39

NA 4.281 1.239 5.633 6.422

159 264 268 275 292

-6.8 -5.77 -7.04 -7.14 -7.1

7.74 7.64 7.83 7.74 7.77

1.01 4.54 4.01 3.24 4.02

NAb

NAb

306

NAb

NAb

NAb

Cited from ref 16.

b

Not applicable.

FIGURE 1. LiP-catalyzed removal of E2 at different pH values and H2O2 concentrations. Experimental conditions: (A) [E2] ) 10 µM; [LiP] ) 0.02 U/mL, [H2O2 ] ) 0.3 mM, pH controlled by 10-mM CPBS; (B) [E2] ) 10 µM; [LiP] ) 0.02 U/mL, pH ) 4.6 (10-mM CPBS). Error bars represent standard deviations (n ) 3). properties of the estrogen molecules and VA were computed and are described in Supporting Information III. These descriptors were used in seeking and establishing relationships with their enzymatic reaction behaviors.

Results and Discussion LiP-Mediated E2 Removal at Varying Reaction Conditions. Our experiments revealed that LiP mediates effective transformation of E2 in aqueous solution, and the data of E2 removal in reactions at varying pH and H2O2 concentrations are displayed in Figure 1. The performance of LiP appeared to be pH-dependent, with an optimum around pH 4, as shown in Figure 1 A. Effective E2 removal can still be achieved at pH 7, although the efficiency was reduced to about one quarter of that at pH 4. Figure 1B shows that 100% removal of 10-µM E2 was achieved with 0.02-U/mL LiP and 0.05-mM H2O2 at pH 4.6, and any increase of H2O2 resulted in reduction of E2 removal. Such a behavior was consistent with the early finding that excess H2O2 inactivated LiP (17, 20). It was proposed that the pH-dependency of LiP performance, as shown in Figure 1A, may be attributable to the presence of an exchangeable proton that plays a critical role in LiP catalysis (14). Because the pH-dependency of LiP seems to be largely attributable to its own characteristic instead of the substrates’ (14, 27, 28), the pH-dependence behavior observed with E2 may be applicable to other estrogens. Products of LiP-Mediated E2 Reaction. We have attempted to identify products formed in the LiP-mediated E2 reactions using Mass Spectrometry. The mass spectrum that we chose to show in Supporting Information IV (Figure S3) was taken on a sample that was prepared under a relatively mild reaction condition (i.e., 19-µM E2, 10-µM H2O2 and 0.01U/mL of LiP). A careful comparison of this spectrum with those of the two blank samples (also shown in Figure S3) indentified two m/z peaks (268.8 and 541.0) that appeared for the reaction sample but not for the blank samples. These two peaks likely correspond to the molecular ions of two reaction products, estrone (MW ) 270) and a dimer of E2 (MW ) 542). The molecular ion of E2 (m/z ) 270.7) was also present in the spectrum of the reaction sample, reflecting incompleteness of the reaction. When samples were prepared at stronger reaction conditions (e.g., 0.02-U/mL of LiP), E2 did not show in the mass spectrum, nor did estrone, indicating the intermediate nature of estrone. The molecular ion of an E2 trimmer (m/z ) 811.0) can also be identified in samples 376

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FIGURE 2. Correlation between Michaelis constant (KM) and molecular volume (VM).

FIGURE 3. LiP-catalyzed removal of E2 in systems containing different concentrations of veratryl alcohol. [E2] ) 30 µM; [LiP] ) 0.006 U/ml, [H2O2] ) 0.01 mM, pH ) 4.6 (10-mM CPBS); Reaction time ) 30 min. Error bars represent standard deviations (n ) 3). that were prepared under stronger reaction conditions. Taken together, the mass spectrometry strongly suggests that E2 tends to polymerize under LiP catalysis, likely via oxidative coupling that has been observed for the reactions mediated by horseradish peroxidase and laccases (29). It has been shown that such enzyme-mediated polymerization can effectively reduce the estrogenicity of parent estrogens (5). Enzymatic Reaction Kinetics and Possible Binding Site for Estrogens. Enzymatic reaction kinetics were evaluated by the “initial reaction rate” technique as described in the Experimental Section, i.e., the reaction rate of each substrate was assessed at varying initial substrate concentrations within a sufficiently short time so that the pseudofirst-order rate behavior of the enzymatic reaction can be captured. The reaction rates thus obtained were plotted against the corresponding initial concentrations of the substrate as shown in Figure S4 of Supporting Information V. Fitting the data to Michaelis-Menten equation allowed determination of the Michaelis constant KM and the maximum reaction rate rmax. The catalytic rate constant kCAT was computed based on rmax as described in Supporting Information III. The KM

FIGURE 4. Removal of E2 over time in LiP-mediated reaction systems variously containing VA and/or NOM, including a reaction system that did not contain VA or NOM; a system containing NOM at 5 mg (TOC)/L; a system containing 1-mM VA; and a system containing both 5 mg/L NOM and 1-mM VA. Experiment conditions: [E2] ) 19 µM; [LiP] ) 0.0065 U/mL, [H2O2] ) 0.01 mM, pH ) 4.6 (10-mM CPBS). Error bars represent standard deviations (n ) 3).

FIGURE 5. Rate of VA conversion in systems containing NOM at various levels. [NOM] ) 0, 1, 5, or 10 mg (TOC)/L, [LiP] ) 0.0067 U/ ml, [H2O2] ) 0.3 mM, [VA] ) 1.67 mM. and kCAT values thus obtained for each studied substrate are tabulated in Table 1, along with the literature data for VA. It is very interesting in Table 1 that KM values of the estrogens, although varying from each other, are all much smaller than that of VA by orders of magnitude. KM essentially represents the affinity between the substrate and the enzyme, with a low KM value corresponding to a high affinity. This suggests that the estrogens bind to LiP much more strongly than VA does. The substantial difference in KM values for estrogens and for VA led us to hypothesize that the active binding site on LiP for estrogens is different from that for VA. There are two widely accepted active binding sites on LiP; one is located within the so-called heme cavity and the other on LiP surface at or near Trp171 (14, 16, 23-25). With the help of site-directed mutation, researchers were able to prove that the binding site for VA was the one located on LiP surface (16, 23-25) and there existed a long-range electron transfer channel between heme and this surface site (23-26). We hence postulated that the heme cavity was most likely the binding site for the estrogens. This appears to explain the much smaller KM values of estrogens than those of VA,

because the binding force between substrate and enzyme tends to be much stronger within a cavity site than at a surface site simply because of geometric effect. To further investigate the binding of estrogens, we calculated several molecular descriptors of the chemicals, listed in Table 1, and attempted to search for the relationship between KM and the molecular structural characteristics of the estrogens. A linear relationship between KM and molecular volumes (VM), as shown in Figure 2, was the only significant relationship (r2)0.9317) that we were able to identify by regression analysis between KM and each of the molecular descriptors listed in Table 1. The strong correlation between KM and VM appeared to support the hypothesis that the estrogens’ active binding site is not on LiP surface but within a cavity, where substrate volume tends to play an important role in binding interactions. It is evident in Figure 2 that VA deviates by far the correlation between KM and VM for estrogens, which further corroborate that VA and estrogens have different binding sites on LiP. It was reported that the LiP binding cavity is located on the distal side of the heme and formed primarily by three amino-acid residues Arg43, VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Phe46, and His47 (24), as indicated in Figure S5 in Supporting Information VI. We further estimated the volume of the cavity to be approximately 306 Å3 by measuring the volume of a cylinder that fits in the cavity as indicated in Figure S5 and associated description in Supporting Information VI. Such a cavity size is slightly larger than the molecular volumes of the estrogens as shown in Table 1. When the measure of the substrate molecule is close to that of the cavity, it is reasonable that the binding would be impeded more as the substrate size increases, which appears to be the case for the estrogens as indicated by the positive correlation between KM and VM. Influence of VA and Natural Organic Matter on LiPMediated E2 Removal. The experimental results shown in Figure 3 indicate that the presence of VA enhanced the removal of E2, which can be attributed to the ability of this compound to protect LiP from inactivation and to enhance LiP reactivity (20, 21, 26, 30-32). Accepted theory regarding the mechanism of LiP-mediated oxidation consists of three steps as depicted in Figure S1 of the Supporting Information. First, LiP is oxidized by H2O2 to form compound I. Compound I obtains an electron from the substrate to form compound II, which obtains a second electron from another substrate molecule to return to the native enzyme. However, a side reaction exists that converts compound II to an inactive enzyme state compound III, leading to enzyme inactivation (31, 32). VA can protect LiP from inactivation via two ways; the first is to help the reduction of compound II to complete the catalytic cycle, thus suppressing the side reaction, and the second is that VA directly interacts with compound III to return it to the native enzyme state (31, 32). Practically, VA holds the LiP activity longer during the reaction, which in turn benefits the removal of other substrates. In addition to this protection effect described above, it is also known that VA is oxidized, during LiP catalysis, to form a cation radical (VA. +), which can spin coupled to the heme iron and form a LiPII-VA. + complex that has a higher redox potential than uncomplexed LiPI and LiPII, thus enhancing the enzyme reactivity. NOM is ubiquitous in nature and is known to be able to affect various environmental processes. Little is known concerning the influence of this species on the behavior of enzymes of environmental significance, such as LiP. To better understand the behavior of this enzyme under real conditions, the removal of E2 mediated by LiP was further investigated in the presence of 5 mg/L (in TOC) NOM. As shown in Figure 4, 5 mg/L NOM by itself had little effect on the transformation of E2. When 1-mM VA was present without NOM, the transformation was significantly accelerated, requiring only ∼20 min to achieve complete removal of 19 µM E2. However, the time required to achieve complete E2 removal increased to 1 h in the presence of both 1-mM VA and 5 mg/L NOM. This result led us to postulate that NOM may be able to compete with VA for the active site located on LiP surface so that VA’s enhancement effect on LiP performance was impaired in the presence of NOM. Therefore, as shown in Figure 4, E2 transformation was significantly increased in the presence of VA due to its effect on LiP performance depicted in the above paragraph; while, in the presence of both VA and NOM, E2 transformation was less than that in the presence of VA only, because NOM competed with VA and reduced its enhancement effect on LiP. A schematic representation of the postulated interactions among VA, NOM, and E2 during LiP catalysis is provided in Figure S6 in Supporting Information VII The competition between NOM and VA was also observed when the transformation rate of VA by LiP was examined in the presence of NOM at different concentrations. VA is oxidized to vertryl aldehyde, which has a characteristic absorbance band at 310 nm. Hence, the absorbance of the reaction solution at 310 nm can be used as a surrogate to 378

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trace the transformation of VA. Figure 5 shows the data of VA conversion over a time period of 7 min in systems containing NOM at different levels. It is clear in Figure 5 that the presence of NOM effectively inhibited the formation of veratryl aldehyde, and the inhibition was quantitatively dependent on the NOM concentration. The conversion of VA was reduced by approximately one-half when NOM is 5 mg/L, and was completely suppressed when NOM level increased to 10 mg/L. This suggests that NOM may have a fairly strong affinity to LiP at the site where VA binds, thus reducing VA reaction and its associated enhancement effect on LiP performance. Considering VA is excreted by fungi along with LiP to mediate the catalytic activity, NOM is an important factor that can affect the overall performance of the enzyme in natural and engineered systems.

Acknowledgments This work was supported in part by U.S. EPA STAR Grant G6M10518 and by HATCH funds. The content of this work does not necessarily represent the views of the funding agencies. L.M. thanks the China Scholarship Council for supporting him as an exchange student to participate in this study at UGA. We are grateful to Ms. Mary Flynn’s help with editing the manuscript.

Supporting Information Available (i) Schematic depiction of LiP catalysis; (ii) Molecular structures of estrogens; (iii) Additional description of certain experimental procedures; (iv)Mass spectra of the products resulting from LiP-mediated E2 reactions; (v) MichaelisMenten curves for LiP-catalyzed estrogen reactions; (vi) A schematic model of the heme cavity in LiP; and (vii) Postulated interactions among E2, VA, and NOM during LiP catalysis. This information is available free of charge via the Internet at http://pubs.acs.org.

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(24)

(25)

(26) (27)

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