Article pubs.acs.org/biochemistry
Substrate Specificity of Sphingobium chlorophenolicum 2,6Dichlorohydroquinone 1,2-Dioxygenase Timothy E. Machonkin* and Amy E. Doerner Department of Chemistry, Whitman College, 345 Boyer Avenue, Walla Walla, Washington 99362, United States S Supporting Information *
ABSTRACT: PcpA is an aromatic ring-cleaving dioxygenase that is homologous to the well-characterized Fe(II)-dependent catechol extradiol dioxygenases. This enzyme catalyzes the oxidative cleavage of 2,6-dichlorohydroquinone in the catabolism of pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. 1H NMR and steadystate kinetics were used to determine the regiospecificity of ring cleavage and the substrate specificity of the enzyme. PcpA exhibits a high degree of substrate specificity for 2,6-disubstituted app app hydroquinones, with halogens greatly preferred at those positions. Notably, the kcat /KmA of 2,6-dichlorohydroquinone is ∼40fold higher than that of 2,6-dimethylhydroquinone. The asymmetric substrate 2-chloro-6-methylhydroquinone yields a mixture of app 1,2- and 1,6-cleavage products. These two modes of cleavage have different KmO values (21 and 260 μM, respectively), 2 consistent with a mechanism in which the substrate binds in two catalytically productive orientations. In contrast, monosubstituted hydroquinones show a limited amount of ring cleavage but rapidly inactivate the enzyme in an O 2-dependent fashion, suggesting that oxidation of the Fe(II) may be the cause. Potent inhibitors of PcpA include ortho-disubstituted phenols and 3-bromocatechol. 2,6-Dibromophenol is the strongest competitive inhibitor, consistent with PcpA’s substrate specificity. Several factors that could yield this specificity for halogen substituents are discussed. Interestingly, 3-bromocatechol also inactivates the enzyme, while 2,6-dihalophenols do not, indicating a requirement for two hydroxyl groups for ring cleavage and for enzyme inactivation. These results provide mechanistic insights into the hydroquinone dioxygenases.
B
hydroxyl-substituted aromatic ring with a hydroxyl, amino, or carboxylate at the ortho and/or para positions. Interestingly, all of these enzymes appear to share essentially the same type of metal-binding site employed by the EDOs: a single Fe(II) center bound to the protein by a 2-histidine-1-carboxylate facial-capping triad of ligands (or, in some cases, three histidines). This would seem to imply that the factors governing the substrate specificity among these ring-cleaving dioxygenases lie in the active site pocket and the second coordination sphere. The dioxygenases that cleave hydroquinones lacking a carboxylate functional group [hydroquinone dioxygenases (HQDOs)] are unique among ring-cleaving dioxygenases in that their substrates cannot bind to the Fe(II) center in a bidentate fashion. However, they have been studied little, and only five have been reported to date. Three of these are homologues that are 40−50% identical in amino acid sequence: 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA) from the pentachlorophenol degradation pathway of Sphingobium chlorophenolicum ATCC 39723 (Scheme 1A),28−30 chlorohydroquinone 1,2-dioxygenase (LinE) from the γ-hexachlorocyclohexane (lindane) degradation pathway of Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 (Scheme 1B),31 and MnpC, an HQDO from a putative
acteria use an astonishing range of molecules as carbon sources, a property that has been of interest for use in the bioremediation of xenobiotic compounds, including recalcitrant pollutants such as chlorinated organic compounds.1−5 The study of these pollutant-degrading catabolic pathways has yielded a wealth of novel enzymes with interesting properties. The group of bacterial enzymes involved in the catabolism of aromatic hydrocarbons is one such example that has received considerable attention. In most cases, aerobic catabolism of aromatic hydrocarbons involves the oxidative ring cleavage of a catechol. The substrate specificity of these catechol dioxygenases can limit the range of aromatic hydrocarbons that can be utilized by a particular catabolic pathway, in part because of the buildup of toxic dead-end metabolites.3 There are two types of catechol dioxygenases: the Fe(III)-dependent intradiol dioxygenases (IDOs), which cleave the C−C bond between the vicinal hydroxyl groups, and the Fe(II)-dependent extradiol dioxygenases (EDOs), which cleave the C−C bond adjacent to the vicinal hydroxyl groups.6−10 Extensive studies of the EDOs using a range of experimental approaches (including structures of trapped intermediates) have yielded a generally accepted mechanism6,7,10−12 supported by density functional theory calculations.13,14 In recent years, ring-cleaving dioxygenases that work on noncatecholic substrates, including salicylates and gentisates,15−21 homogentisate,22,23 2-aminophenols,24−27 and hydroquinones (that lack a carboxylate functional group), have been described.28−34 All of these substrates consist of a © 2011 American Chemical Society
Received: June 2, 2011 Revised: August 23, 2011 Published: August 26, 2011 8899
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the regiospecificity of the enzyme and the tautomeric states of the cleavage products. The results provide insights into the mechanism of ring cleavage.
Scheme 1. Ring-Cleavage Reactions Catalyzed by the Known HQDOs
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EXPERIMENTAL PROCEDURES Protein Production. The expression and purification of PcpA were performed by the method of Xun and co-workers 30 with the modifications described previously.35 Preparation of Potential Substrates and Inhibitors. 2,6-Dibromobenzoquinone was synthesized by the method of Ungnade and Zilch.51 The identity of this compound was confirmed by 1H NMR (CDCl3): δ 7.33 (s). 2-Chloro-6methylhydroquinone was synthesized by the method of Burton and Prail, with the following additional purification steps.52 The product of the first step (1,4-diacetoxy-2-chloro-6-methylbenzene) was purified by silica gel column chromatography. The identity of this compound was confirmed by 1H NMR (CDCl3): δ 6.99 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 2.2 Hz, 1H), 2.30 (s, 3H), 2.22 (s, 3H), 2.13 (s, 3H). The final product was recrystallized from hot dichloromethane. The identity of the product was confirmed by 1H NMR (CDCl3): δ 6.71 (d, J = 2.8 Hz, 1H), 6.58 (d, J = 2.8 Hz, 1H), 2.26 (s, 3H). The purity of both compounds was determined to be >98% by both thin layer chromatography and 1H NMR. Chlorobenzoquinone was purchased from Alfa Aesar and repurified by silica gel column chromatography. All other compounds were purchased commercially, were reagent grade, and were used without further purification. Kinetic Studies. The rate of the enzymatic reaction was determined by measuring product formation by a spectrophotometric assay developed by Xun and co-workers30 with the modification (addition of imidazole) described previously.35 The assay was performed on a Cary 50 Bio or Cary 5000 (for the O2 dependence experiments) UV−visible spectrophotometer (Varian). In some cases, the hydroquinones were formed by in situ reduction of the substituted benzoquinone with NaBH4. This was done with 2,6-dichlorohydroquinone, 2,6bromohydroquinone, and 2,6-dimethylhydroquinone. To ensure that the absorbance change of the reaction mixture was within a measurable range, either a 1.0 cm path length cuvette with a 1.0 mL reaction volume or a 5.0 cm path length cuvette with a 2.0 mL reaction volume was used. The 1.0 cm path length cuvette was used for initial screening of possible substrates, for all of the inhibitor studies, for the substrate dependence steady-state kinetics of 2,6-dimethylhydroquinone, and for the O2 dependence steady-state kinetics. In these studies, 5−10 μL of 85−110 μM PcpA was used. The 5.0 cm path length cuvette was used for the substrate dependence steady-state kinetic studies of 2,6-dichlorohydroquinone, 2,6dibromohydroquinone, and 2-chloro-6-methylhydroquinone, in which 2.5 μL of 110 μM PcpA was used. For the initial screening of substrates, both high (200−400 μM) and low (10−60 μM) concentrations of the compounds in question were tested. Air-saturated 20 mM potassium phosphate buffer (pH 7.0) was used throughout. All reactions were performed at 23 °C. Because of the use of NaBH4 for in situ reduction of the substituted benzoquinones to the corresponding hydroquinones, bubbles of H2(g) formed during the course of the reaction, thereby causing a small baseline shift in the absorbance spectrum. To account for this absorbance shift, the baseline absorbance, measured at 380 nm, was subtracted from the product absorbance at each time point.
m-nitrophenol degradation pathway of Cupriavidus necator JMP134.34 The two other HQDOs share no sequence identity with PcpA but are >60% identical in sequence with each other and cleave unsubstituted hydroquinone: HapCD from the 4hydroxyacetophenone degradation pathway of Pseudomonas f luorescens ACB32 and PnpC1C2 from the p-nitrophenol degradation pathway of Pseudomonas putida DLL-E4 33 (Scheme 1C). Thus, similar to the EDOs, which occur in two different classes (type I and type II),8,9 there are two distinct structural classes of HQDOs. PcpA and its homologues (LinE and MnpC) and the type I EDOs are members of the “vicinal oxygen chelate” (VOC) superfamily, as first recognized by Xu et al.29 However, our experimentally verified structural model of PcpA demonstrated that the four copies of the βαββββ structural motif of the VOC superfamily are arranged differently in this enzyme and that residues His11, His227, and Glu276 are likely the ligands to the Fe(II) center.35 A remarkable property of PcpA and LinE is their ability to cleave chlorinated substrates. This contrasts with EDOs, which are known to be subjected to mechanism-based inactivation by chlorinated substrates.36−39 However, whether PcpA and LinE exhibit an actual preference for chloro substituents or merely tolerate chloro substituents has not been previously established, because very little about the substrate specificity of these enzymes has been reported. Another HQDO, HapCD, has a higher activity with methylhydroquinone than it does for its native substrate, hydroquinone, and a lower activity toward chlorohydroquinone and other halogenated hydroquinones. 32 Understanding how a naturally occurring ring-cleaving dioxygenase can successfully utilize chlorinated substrates is an important area for study. Considerable effort has been spent in identifying EDOs capable of cleaving chlorinated substrates39−46 and engineering EDOs with improved abilities to cleave chlorinated substrates and/or reduced sensitivity to inactivation;47−50 however, success on the latter front has been limited. In this work, we investigated the range of molecules that can act as substrates and inhibitors of PcpA, including structural variants of the physiological substrate (2,6-dichlorohydroquinone) and substrates of other ring-cleaving dioxygenases. Steady-state kinetic studies were used to establish the substrate specificity of the enzyme as well as the mode and strength of inhibition of various inhibitors. 1H NMR was used to determine 8900
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For the initial screening of possible substrates, the spectrophotometric assay was conducted with a range of compounds, and changes in the UV−visible absorbance spectrum were monitored. For each compound tested, a control experiment in the absence of PcpA was conducted to ensure that enzymatic activity was the source of any spectral changes. The appearance of a new transition in the PcpAcontaining reaction but not in the control was determined to be representative of ring-cleavage activity. Molecules that were determined to be cleaved by PcpA were further characterized by steady-state kinetics. The initial velocities were measured, divided by the enzyme concentration used, and plotted versus substrate concentration, and the Michaelis−Menten equation was fit to the data with KaleidaGraph (Synergy) to yield the app app KmA and kcat values in air-saturated buffer (O2 concentration of 267−275 μM). The dependence of the O2 concentration on the rate in the presence of high concentrations (200 or 300 μM) of the substrates was determined by the same spectrophotometric method, and the Michaelis−Menten equation was fit to the data to yield the apparent KmO2 and kcat values. The O2 concentrations in the buffers were determined with an O2-sensitive electrode (Hansatech) in separate control experiments without PcpA. For the initial screening of inhibitors, the spectrophotometric assay was conducted with the compound in question at 500 μM and the native substrate at 50 μM, and the initial velocity relative to that without inhibitor was calculated. Detailed steady-state kinetic characterization was performed on three of the strongest inhibitors. Likewise, the kinetics of the monosubstituted hydroquinones were analyzed by treating them as inhibitors of 2,6-dichlorohydroquinone. For each inhibitor, the initial velocities were determined with five or six different substrate concentrations at each of three or four different inhibitor concentrations (and in the absence of inhibitor). The inhibition constant, KI, was determined by performing a global fit to the data set using the Solver function of Excel (Microsoft). Only the value of KI was allowed to float. The inhibitor studies were performed in the 1.0 cm path length cuvette, which was more convenient to use and required less material, whereas determination of the kinetic parameters of 2,6-dichlorohydroquinone required use of the 5.0 cm path length cuvette because of the low molar absorptivity of the app product and the low KmA for this compound. Use of only 1.0 cm path length data consistently led to a large overestimate of app app KmA . Thus, in the fitting of the inhibitor data, the value of KmA determined from the 5.0 cm path length data was kept fixed. The uncertainty in KI was estimated by a jackknife procedure53 app and propagated with the uncertainty in KmA by standard methods. Enzyme inactivation with different compounds was assessed by several methods. (1) The curvature during product formation was directly observed in the spectrophotometric assay. Conditions under which the enzyme was completely inactivated before the substrate was consumed were used (0.46 μM enzyme and 50 μM substrate for the monosubstituted hydroquinones and 0.146 μM enzyme and 200−300 μM substrate for the ortho-disubstituted hydroquinones), and this was used to estimate the partition ratio:38
(1)
(2) PcpA was incubated both with and without the compound in question in either air-saturated buffer or O2-free buffer, followed by addition of substrate (and more air-saturated buffer, in the latter case). (3) The curvature in the product formation of the native substrate when the compound in question was used as an inhibitor was fit to the equation (2)
to yield the rate of inactivation, js (Pt, Pi, and P∞ are the amounts of product formed at time t, at the start of the assay, and at time ∞, respectively). The apparent rate constant of app inactivation, jinact , and the apparent binding constant of app inactivation, Kinact , were obtained by performing a global fit of the values of js as a function of the inactivating compound concentration, [I], at different substrate concentrations, [A], with Solver:
(3)
This procedure has been reported previously be Eltis and coworkers.38 Product Determination. PcpA reaction products were identified in two different ways. First, the aqueous reaction mixture (in an 86% H2O/10% D2O/4% CD3OD mixture) was directly characterized by 1H NMR spectroscopy, with sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS) used as the chemical shift standard. For these reactions, PcpA was buffer exchanged into 20 mM potassium phosphate buffer (pH 7.0) and reconstituted with iron(II) anaerobically (without the use of imidazole) in a glovebox. The substrate concentration was 2.0 mM. Second, the aqueous reaction mixture was acidified to pH 40-fold above KmA ) for 2,6-diClHQ and 2,6-diBrHQ, the app direct fit of the O2 dependence data with the Michaelis−Menten equation yields the true value of kcat, and the KmO should be approximately equal to 2 app app the true KmO2 (a 10-fold difference between KdA and KmA would give a difference between KmO and K of 1 h for 2-Cl-6-MeHQ and 2,6-diMeHQ). Given the length of time required for inactivation, these values should be viewed with some caution and may be lower limits on the actual partition ratios. In particular, the dihalo-HQs air-oxidize to the corresponding benzoquinones on this time scale, and the presence of minute quantities of the benzoquinone was itself found to reduce the rate of ring cleavage. The partition ratios for the disubstituted HQs were all fairly similar. From the 8905
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were plotted as a function of monosubstituted HQ concentration at different 2,6-diClHQ concentrations and fit with a competitive inhibition-type equation (eq 3), in which 2,6-diClHQ inhibits the enzyme inactivation caused by one of these compounds. The resulting apparent rate app constants of inactivation, jinact , are listed in Table 3. Qualitatively, MeHQ showed less curvature, thus precluding an accurate fit to the js data. Partition constants were app estimated, and from the partition constants and jinact values, app app app kcat and in turn kcat /KmA were estimated for ClHQ and app BrHQ. The kcat values were only somewhat lower than those for the dihalo-HQs, and the specificity constants were between those of the dihalo-HQs and 2,6-diMeQ. The most notable difference between the dihalo-HQs and ClHQ and BrHQ is that for the latter substrates the partition ratio was app ∼10-fold lower and, correspondingly, jinact was ∼10-fold higher. Thus, overall, it appears that there is an interaction between the ortho substituents of the substrate and the active site that app app significantly affects kcat /KmA and modestly affects kcatapp, indicating that this interaction leads to a much greater specificity for HQs with halogen substituents versus methyl substituents. Furthermore, the removal of one ortho substituent greatly increases the rate of inactivation. Kinetic Parameters of O2. The O2 dependence on the initial velocities for 2,6-diClHQ, 2,6-diBrHQ, and 2,6-diMeHQ at high substrate concentrations is shown in Figure 3A, and the resulting kinetic parameters are listed in Table 4. The kcat/KmO2 values were remarkably similar for these three symmetric substrates, and both the KmO2 and kcat values for these substrates shared the same trend: 2,6-diClHQ > 2,6-diBrHQ > 2,6diMeHQ. In the presence of saturating amounts of 2-Cl-6-MeHQ, the ratio of cleavage products was strongly dependent on O 2 concentration. Thus, the initial rates of the increase in absorbance at 250 and 314 nm displayed different dependencies on O2 concentration (Figure 3B). Several mechanisms were considered in which a single organic substrate could bind to the enzyme to yield two different products. A mechanism consistent with the observation that the product ratio is invariant with respect to substrate concentration but varies with O2 concentration is shown in Scheme 3. This yields the following equations for the initial rates of formation for the two products
mechanism in Scheme 3 (see the Supporting Information for the derivations and for the definition of these coefficients). At a Scheme 3. Mechanism by Which 2-Cl-6-MeHQ Binds to PcpA in Two Different Orientations Yielding Two Different Products
fixed [O2], because the denominator terms of V and V′ are identical and the numerator is first-order in [A], then the ratio of product formation as function of [A] is constant (in agreement with experiment) and the initial rates should follow Michaelis−Menten kinetics with the following equations for app app kcat and KmA :
(6)
(7)
(8)
At a fixed [A], the initial rates do not follow true Michaelis− Menten kinetics. However, if the terms in eqs 4 and 5 that are second-order in [O2] are small, the O2 dependence will still show saturation behavior that can be fit to a Michaelis−Menten app type of equation; however, the resulting kcatapp and KmO values 2 cannot be related back to the Nx and Dx terms. The ratio of the initial rates of formation of the two products as a function of [O2] is (9)
consistent with the experimentally observed nonlinear O 2 dependence on this ratio. Importantly, a mechanism in which the asymmetric substrate is in rapid equilibrium between the two binding modes without dissociating from the enzyme does not yield the observed dependence of the O2 concentration on the product ratio, nor does a mechanism in which the substrate binds in a single orientation and then partitions between two different ring-cleavage pathways. However, there may be other mechanistic possibilities consistent with this observation, such as one binding mode leading to partial enzyme inactivation. This behavior in which a single substrate yields two different app app products with the same KmA values but different KmO values 2 has been observed before in a ring-cleaving dioxygenase. It was first reported in Pseudomonas arvilla catechol 1,2-dioxygenase (an IDO) with the substrate 3-methylcatechol, which led to a mixture of 1,2- and 2,3-cleavage products, although a detailed analysis of the kinetics was not performed.62 The transition at 314 nm arises from just the 1,6-cleavage product, while the transition at 250 nm arises from both products. Therefore, the initial rates of increase at both wavelengths were simultaneously fit with two Michaelis−Menten
(4)
(5)
where [A], [O2], and [Et] are the concentrations of the HQ substrate, O2, and enzyme, respectively, and the Nx and Dx coefficients are functions of the rate constants from the 8906
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app app equations (Figure 3, solid lines) with different kcat and KmO 2 values and with the reported molar absorptivity at 250 nm of the 1,2-cleavage product (2-methylmaleylacetate)54 and the estimated molar absorptivities of the 1,6-cleavage product (chloromaleylacetone) at both 250 and 314 nm calculated app app above. This yielded the kcat and KmO values for 1,2- and 1,62 app cleavage listed in Table 3. Notably, the kcat values are similar, app while the KmO2 values differ ∼10-fold. Although there is considerable uncertainty in the molar absorptivity of chloromaleylacetone at 250 nm, even a 50% error in this value app changes the KmO values by
