Biometals (2015) 28:861–868 DOI 10.1007/s10534-015-9871-7
Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site Yu Pang . Wanjun Lan . Xuelei Huang . Guanke Zuo . Hui Liu . Jingyan Zhang
Received: 5 September 2014 / Accepted: 18 June 2015 / Published online: 24 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe3? ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe3? is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe3? ions, two residue clusters of OxOx were assigned as potential Fe3? binding sites, the mechanism of the inhibition of Fe3? was delineated. Importantly, the residues that interact with Fe3? ions are involved in the substrate orienting based on
Electronic supplementary material The online version of this article (doi:10.1007/s10534-015-9871-7) contains supplementary material, which is available to authorized users. Y. Pang W. Lan X. Huang G. Zuo H. Liu J. Zhang (&) State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China e-mail: [email protected]; [email protected]
computer docking. Consequently, the interaction of OxOx with Fe3? highlights insight into substrate binding site in OxOx. Keywords Oxalate oxidase Fe3? ion Inhibition Computer modeling Substrate binding site
Introduction Oxalate is a toxic compound for human primarily obtained through a diet of oxalate rich plants. High oxalate concentration can cause hyperoxaluria, kidney stones, and cardiological diseases (Hodgkinson 1977; Williams and Wandzilak 1989). Biological research on oxalate degrading enzymes is thus attracting for the potential applications in the oxalate related diseases. Oxalate oxidase (OxOx, EC 1.2.3.4) is one of typical oxalate degrading enzymes, it catalyzes the oxidative cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide (Woo et al. 1998). It is a hexameric enzyme, each subunit possess a barrel structure and a manganese ion locates inside the barrel (Requena and Bornemann 1999). The manganese ion at the active site is bound by four residues (His88, His90, Glu95, and His137) together with two water molecules forming an octahedral coordination environment (Woo et al. 2000). Oxalate oxidation is occurred at the manganese centers. In spite of the successful structural characterization of OxOx in
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yeast, details of its catalytic mechanism remain unclear. It has long been believed that during the catalytic cycle oxalate anion interacts with the MnII center in monodentate mode, and one dioxygen molecule displaced the two coordinated water molecules. However, recently, Whittake et al. proposed that in the resting state, manganese is in oxidation state MnIII, and substrate binds to OxOx forming an enzyme-oxalate complex based on EPR and redox modification studies (Whittaker et al. 2007). Vicky et al. modified OxOx using the specific chemical modifiers to identify surface amino acids that are essential for enzyme activity. They concluded that one carboxylate and one lysine on the per monomer surface was critical for enzyme activity (Kotsira and Clonis 1998). A recent study on OxOx using the complex of the enzyme and substrate analogue glycolate implied that the manganese ion at the active site serves two functions: to organize the substrates (oxalate and dioxygen) and to reduce transiently dioxygen (Opaleye et al. 2006). Several residues are believed critical in orientating the substrates and reaction intermediates for catalysis. There are apparently several key issues of the OxOx mechanisms that are still not well understood, such as substrate binding site, and binding mode etc. In this work, to delineate the substrate binding site, the interaction of Fe3? ions with OxOx was investigated. Effects of various metal ions on the enzyme catalysis, including Na?, K?, Ca2?, Cu2?, Pb2?, and Fe2?were previously investigated to identify the active site before the crystal structure of OxOx was obtained. Monovalent Na?, K? and divalent Mg2?, Zn2?, Cu2?, and Ni2?ions have no effect on the enzyme activity (Kotsira and Clonis 1998). In contrast, earlier work reported that 1 mM of Pb2? marginally activated OxOx, and Pb2? inhibited the enzyme activity at a low concentration (Sugiura et al. 1979). And some reported that high concentration of Fe2? and Fe3? inhibited OxOx almost completely. Whereas Ca2?, Cu2?, and Pb2? stimulated the activity of OxOx at different extents. The mechanism of inhibition or activation to OxOx by metal ions remains unrevealed. However, the effect of metal ions on the activity of OxOx apparently is closely related to the some steps of the enzyme catalysis. Combining with biological methods, spectroscopic method, and computer modeling, interaction of Fe3? with OxOx is systematically studied. We assigned two groups of the
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residues of OxOx that interact with Fe3?, and are critical in orienting substrate binding to the enzyme, hence indicates the substrate binding site.
Materials and methods Materials The barley seeds were provided by Institute of Agriculture, Shanghai Academy of Agriculture Sciences. CMSepharose Fast Flow and Sephacryl, S-200 resins are products of Amersham Biosciences. Horseradish peroxidase (HRP) was purchased from Yuamju Biotech. CO, Lit. Analytic grade oxalic acid, hydrogen peroxide, phenol red, CuSO4, ZnSO4, FeSO4, Fe(NO3)3, MnCl2, CaCl2 were purchased from the Sinopharm Chemical Reagent CO., Ltd. And prepared freshly before the experiment. Enzyme purification OxOx was purified from 10 days old barley roots using the reported method with modifications (Satyapal and Pundir 1993). Frozen roots were ground with chilled deionized water (1:3) at zero degree. The resulting mixture was filtered by cheese cloth first; the filtrate was centrifuged for 30 min at 4 °C giving a crude extract. Solid ammonium sulphate was added to the crude extract to give a final 80 % saturation, and was maintained at 4 °C overnight. The resulting mixture was centrifuged for 30 min and the supernatant was discarded. The sediment was dissolved in distilled water, and the solution was dialyzed against Milli Q water for 16 h. The dialyzed solution was loaded onto CM-Sepharose column (U 2.6 9 30 cm) pre-equilibrated with 0.01 M,pH 4.5 sodium acetate buffer. The enzyme was eluted with a linear gradient of NaCl (0–0.6 M) in the same buffer. The enzyme fractions were pooled and concentrated to *500 lL. Because OxOx is heat stable (Kotsira and Clonis 1997), the pooled fraction from CM-Sepharose column was heat treated (50 °C for 6 min) and centrifuged to remove precipitated protein. The resulting solution was applied on a Sephadex G-200 column (U 1.5 9 75 cm), which is previously equilibrated with 0.05 M sodium succinate buffer (pH 5.0). The active fractions were pooled and concentrated.
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The purity of the concentrated enzyme was checked by SDS-PAGE and stored at -20 °C. Activity assay OxOx can convert oxalate into hydrogen peroxide and carbon dioxide, and the activity of the enzyme was assayed by monitoring the H2O2 using two colorimetric methods, phenol red-HRP (Zuo et al. 2010) and DMA-MBTH methods (Zuo et al. 2010), and the model compound method that developed by our group (Zuo et al. 2010). For DMA-MBTH method, some metal ions may interfere with the assay (Nakano et al. 1985), therefore in this work, phenol red-HRP method was employed mainly. Generally, the reaction mixture (0.2 mL) contained OxOx (0.1 lM), oxalic acid (2 mM) in 20 mM, pH 3.5 sodium succinate buffer was incubated at 40 °C for 5 min, 0.4 mL of sodium succinate buffer (50 mM, pH 5.0) containing phenol red (0.1 mM) and 6.25 U of HRP were then added to detect the reaction product H2O2. The mixture was then incubated at 30 °C for 15 min, 0.1 mL of 5 M NaOH was added to stop the reaction, and the absorbance of the mixture at 610 nm is proportional to the H2O2 concentration in the reaction system. The amount of H2O2 generated during the reaction was calculated according to the standard curve. The effect of metal ions was measured based on the method of enzyme activity assay including metal ions. OxOx was first incubated with the solution of different metal ions for 10 min at 4 °C to ensure metal ions completely bind to OxOx. Microscale thermophoresis (MST) MST was measured with a Nano Temper Monolith NT.115 system (Germany). The concentration of OxOx was 100 nM. OxOx was labeled by Monolith NTTM protein labeling kit with NT-647. The labeled proteins were added to the capillaries (Nano Temper Technologies GmbH) with different concentrations of Fe3? or Ca2?. The binding process of OxOx and Fe3? is characterized by:
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FB =
¼
½AB ½B
½A þ ½B þ KD
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½A þ ½B þ KD Þ2 4½AB 2½B
FB is described by: Fnorm = (1 - FB)Fnorm,unbound ? (FB)Fnorm,bound; Fnorm,unbound: normalized fluorescence of the unbound OxOx; Fnorm,bound: normalized fluorescence of the bound state (Seidel et al. 2013). Circular dichrosim (CD) and UV–Visible measurements A JASCO J815 spectropolarimeter (Jasco International Co. Ltd., Japan) equipped with a Jasco temperature controller (model PTC-423S) and controlled by a PC was used for all circular dichrosim measurements at 22 °C. Milli Q water was used to avoid the interference of the buffer to the CD spectra. A quartz cell with 1 cm path length was used. Each spectrum was averaged from five successive accumulations at a scan rate of 50 nm/min. UV–Visible absorption spectra of the samples were obtained with a Cary 50 spectrophotometer. Computer modeling and docking The crystal structures of OxOx (PDB code 1FI2), were downloaded from http://www.rcsb.org/pdb/ for computer modeling. The surface potential of OxOx was generated by Ds Visualizer 15 program (Accelrys, Inc.). The amino acid residues that interact potentially with metal ions were screened manually within the negatively charged surface area of the OxOx. The docking of the substrate with OxOx was carried out using OxOx with glycolate complex (PDB code: 2ET1) as a template. The docking was performed at ˚ area. the glycolate binding site with a diameter of 10 A The Glide software (Schrodinger, Inc.) was used on linux workstation for the model building.
Results and discussion
A þ B AB
Purification of OxOx
A Fe3? or Ca2?, B OxOx, AB bound complex of OxOx and Fe3?/Ca2?. The equilibrium dissociation constant KD is obtained by fraction bound (FB):
OxOx was purified to homogeneity from barley roots as described in the experimental section. All the
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procedures for isolation and purification of the enzyme were carried out at 4 °C. The overall purification achieved 580-fold activity with a yield of 20.81 % as summarized in Table S1. The purified OxOx exhibited a single protein band in SDS PAGE as shown in Figure S1. The purified enzyme was stored at -20 °C. Interaction of Fe3?with OxOx The inhibitory effect of metal ions on the activity of OxOx though controversial possibly provides information of substrate binding (Zuo and Zhan 2005). It was reported that many monovalent and divalent metal ions are effective to OxOx activity, we focused primarily on Fe3?. As shown in Fig. 1a, OxOx activity was gradually inhibited with the increase of Fe3? concentration, and was almost completely inhibited with 1 mM of Fe3?, IC50 of Fe3? under the condition is 0.6 mM. To preclude the false inhibition caused by the assay method, the activity of OxOx was measured with different methods complementarily and the results are consisted with each other. However, in the different methods as described in experimental section, the activity of OxOx was evaluated by monitoring catalytic product H2O2 (Mongkolisirikieat and Srisuwan 1987). Since H2O2 can easily react with Fe3? through Fenton reaction (Aplin et al. 2001; Zuo and Zhan 2005), the presence of Fe3? could possibly result in a false inhibition. Control experiments with standard H2O2 samples were therefore performed, and the results clearly indicated the assumption of H2O2 by excess Fe3? in the reaction system was not interfered as displayed in Figure S2. On the other hand, metal
Fig. 1 a Inhibitory effect of Fe3? on the activity of OxOx under different Fe3? and pH. Oxalic acid was 2 mM, and OxOx was 0.010 lM. b Inhibitory ability of Fe3? under different concentrations of substrate. Fe3? concentration was 0.5 mM.
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ions such as Fe3?, Mn2?, Ca2? can react with the native substrate oxalic acid forming metal oxalates to consume some oxalic acid, thus will result in a false low activity of OxOx. However, when excess oxalic acid was used, even with very low concentration of Fe3?, the inhibitory effect was still observed. Thus, the inhibitory effect observed with Fe3? was not due to the substrate lose that caused by the formation of oxalates. Intrinsic OxOx contains manganese ions at the active sites, the replacement of manganese ions also could lead to the loss of activity. To exclude the possibility that Fe3? replace Mn2? at the active site, the activity of OxOx was measured in the presence of metalchelating agent EDTA (3.0 mM) in the reaction system, and the activity is slightly affected, which suggested that manganese ions in OxOx are fairly strong chelated by the amino acids residues at the active site, the replacement of Mn2? by other metal ions is unlikely to occur under the assay condition (Mongkolisirikieat and Srisuwan 1987). The activity of the Fe3? inhibited OxOx can be mostly recovered in the presence of excess of EDTA as shown in Figure S3. Taken these results together, we demonstrate clearly that Fe3? ions can inhibit the activity of OxOx. To further confirm the result, inhibition of Fe3? to OxOx was also assayed with different substrate concentrations. As shown in Fig. 1b, in the presence of Fe3?, the amount of the reaction product H2O2 decreased at the different substrate concentrations. The Lineweaver–Burk plot indicated that the Km was not changed in the presence of Fe3? (Figure S4), indicating that Fe3? ion is a non-competitive inhibitor with Ki of 0.75 mM. This is to be expected, because
c MST analysis of Fe3? binding to OxOx. The assay was carried out in 20 mM sodium succinate buffer with 0.05 % Tween-20, and the concentration of OxOx was 0.1 lM
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Affinity of Fe3? to OxOx To measure the affinity of Fe3? to OxOx, microscale thermophoresis (MST) analysis was performed. MST is based on thermophoresis of protein molecules, that allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The molecule mobility in the temperature gradient is analyzed via fluorescence (Seidel et al. 2013). As shown in Fig. 1c, at pH 3.5, with the titration of Fe3?, the fluorescence of protein was decreased, and the binding of Fe3? was observed, comparing to the experiment with Ca2? under the same condition. However, the binding constant of Fe3? was 2.2 mM, which is relatively high. When pH was increased to 6.0, the binding constant decreased dramatically to 0.49 mM, indicating a stronger binding, which is in consistent with the inhibition assay under the same pH (Fig. 1a). This is definitely not a nonspecific electron static interaction, because control experiment with Ca2? did not show any binding. However, such binding constant is relative high, though similar number was reported for other proteins in the literature (Cassland et al. 2004). Inhibitory mechanism of Fe3? The pH dependence of the binding affinity of Fe3? further confirmed the inhibitory of Fe3? was through interaction with the negatively charged areas on the surface of OxOx as shown in Figure S5. To delineate the mechanism of the inhibitory ability of Fe3?, the effect of Fe3? on the OxOx conformation was first examined with circular dichromism (CD). As shown
4 3+
Fe (0.05mM) OxOX 3+ OxOX+ Fe (0.05mM)
3 2 1 0
-1.0
-1
CD (mdeg)
CD (mdeg)
the substrate oxalic acid is more negatively charged, while Fe3? ions are positively charged, in principle they will not bind at the same site of the enzyme. When the experiment was carried out at pH 6.0, which is higher than pI (4.2) of the OxOx, the inhibitory effect is stronger as showed in Fig. 1a compared with the inhibition measured at pH 3.5 (optimum pH for enzyme catalysis). In the presence of 0.4 mM of Fe3?, only *26 % activity was remained at pH 6.0, while about 48 % activity was maintained at pH 3.5. Apparently, when pH of the reaction buffer is above pI of OxOx, the interaction of Fe3? with OxOx is stronger suggesting that Fe3? ions bind at the negatively charged areas of OxOx.
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-2
-1.5
-2.0
-3 210
220
230
Wavelength (nm)
-4 180
200
220
240
260
280
300
Wavelength (nm)
Fig. 2 CD spectra of the OxOx in the presence of Fe3?. The concentration of Fe3? was 0.05 mM, and OxOx was 0.4 lM
in Fig. 2, there are obvious changes in the CD spectrum of the OxOx in 190–250 nm range upon Fe3? ions binding. The positive band at *195 nm, which is typically for a helix and b sheet, increased, and the negative band at 210 nm decreased, while band at 220 nm increased a little bit. The band at 220 nm shifted 5 nm suggesting that the secondary structure of OxOx was slightly affected by the binding of Fe3? ions. However, there is no change in near UV range 260–300 nm, indicating no changes occurred to the aromatic residues of OxOx upon Fe3? ions binding. CD result provides an evidence that Fe3? ions possibly change the secondary structure of the OxOx, which probably is one of the reasons of its inhibition. Fe3? and substrate binding site On the basis of the intrinsic property of Fe3? ion, its non-competitive inhibition to OxOx under different pH conditions, and the inhibition of Fe3? can not be recovered completely by the presence of excess of EDTA, we thus infer that Fe3? ions most likely interact with OxOx at its negatively charged surface. In fact, there are several large negatively charged domains on the surface of electrostatic potential of the OxOx dimmer and monomer (Figure S5). The most possible targets for Fe3? ions are aspartic acid or glutamic acid resides that exposed on the surface of OxOx. Unfortunately, it is not easy to dock a single metal ion with an enzyme using conventional docking programs, such as Z-DOCK or Swiss DOCK. Hence,
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to support this hypothesis, Fe3? ion was manually docked with the OxOx in the negatively charged surface areas. The criteria for locating Fe3? ions are the spatial distances between Fe3? and oxygen and nitrogen atoms of the surface residues that can potentially form a covalent bond with Fe3? ion. Surfing the negatively charged areas on the surface of OxOx, two potential metal ions binding sites, Glu58 and Asp60 (site I), Asn85 and Gln139 (site II) residues clusters were found. As shown in Fig. 3a, at the site I that located on the wall of the b-barrel of the OxOx, Fe3? is surround by four oxygen atoms from Glu58 and Asp60, and two water molecules nearby (Fig. 3b). Similarly, as shown in Fig. 3c, d, Fe3? can be coordinated by the oxygen and nitrogen atoms from Asn85 and Gln139 (site II) and water molecules nearby. The distances between Fe3? and N, O atoms from the residues at these two sites summarized in Table S2 are similar to the distances that observed in many iron containing enzymes and enzyme model compounds (Woo et al. 1998). Crystal structure of the complex of the recombinant OxOx and substrate analogue glycolate revealed that Asn75, Asn85, and Gln139 might be involved in orienting and stabilizing
of the complex of the enzyme with the substrate or intermediates. Site-directed mutagenesis of the two asparagines in the recombinant OxOx also demonstrated that they are essential for OxOx activity (Opaleye et al. 2006). Interestedly, in the residue clusters we identified (Sites I and II), residues Asn75, Asn85, or Gln139 are all directly or indirectly involved. At the site I, Asn85 binds to water molecule that coordinated to Fe3? (Fig. 3b). Atthesite II, Asn85 was involved in chelating to Fe3? (Fig. 3c). The modeling result illustrated the experimental data that the binding of Fe3? could inhibit the activity of OxOx, and inhibition of Fe3? was via interactions with Glu58 and Asp60, Asn85, and Gln139 residues. However, the above result cannot be further established by site-directed mutagenesis, the attempt to clone OxOx gene and express active OxOx in E. Coli was unsuccessful, probably because of its large size (Cassland et al. 2004). Fortunately, crystal structure of OxOx with glycolate was reported, glycolate was regarded as the substrate analogue (Opaleye et al. 2006). Hence, we docked oxalic acid with OxOx based on the crystal structure of OxOx/ glycolate complex, the binding site of glycolate was
Fig. 3 Two potential Fe3? binding sites on OxOx. a site I (Glu58 and Asp60), c Site II (Asn85 and Gln139). c, d are the ball-stick models of the Fe3? coordination at the Sites I and II,
respectively. The red is oxygen, grey is carbon, and light blue is nitrogen, and yellow is manganese. The crystal structure of OxOx (PDB code 1FI2) was used. (Color figure online)
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Fig. 4 a Docking model of OxOx-oxalic acid complex. Two residues (Asn85 and Asn75) together with H2O orient the substrate oxalic acid and stabilize it through hydrogen bonds. The docking of the substrate with OxOx was carried out using OxOx with glycolate complex (PDB code: 2ET1) as a template.
b Inhibitions of Cu2?, Zn2?, and Ca2? ions to the activity of OxOx. The concentration of all the metal ions is 1 mM. Control is the OxOx alone without any extraneous metal ions. The detailed reaction conditions are described in the experimental section
chosen as a binding pocket for oxalic acid. The docking was carried out by further energy minimization. As shown in Fig. 4a, in the best docking model, oxalic acid locates inside barrel, and is in close proximity to the manganese, hydrogen bonding with two residues Asn85 and Asn75. The bond length of ˚ , Mn–O (H2O) is 2.36 A ˚, Mn–O (oxalic acid) is 2.00 A which are quite similar to those found in manganese complexes (Bacelo and Binning 2006; Mason et al. 2009; Scarrow et al. 1994; Smulevich et al. 1995). The ˚, bond length of O(Asn85)–H(Oxalic acid) is 1.61 A ˚ H(Asn75)–O(Oxalic acid) is 1.97 A, and H(H2O)– ˚ , which are short enough to O(Oxalic acid) is 1.61 A form hydrogen bonds. EPR and redox modification studies had showed that oxalic acid binds to OxOx form an enzyme-oxalate complex (Whittaker et al. 2007). The interactions with Asn85 and Asn75 residues apparently play key role in orienting the oxalic acid to the b-barrel, and close to active site. And more importantly, these residues are also involved in the interaction with Fe3?. When Fe3? binds to OxOx, the interaction of these residues with the oxalic acid was interfered, thus the activity of the OxOx was inhibited. The interaction of OxOx with Fe3? highlights insight into substrate binding site in OxOx. Based on the above results, the Site I that assigned by modeling is apparently not a favorable site comparing to the Site II, that’s probably the reason binding affinity of Fe3? is low (Fig. 1c). Generally, the interaction of metal ions with OxOx is dependent on
the intrinsic properties of the metal ions (Fig. 4b). Under the same concentration, Ca2? ion did show any inhibitory effect, which is consistent with its no binding affinity from MST analysis (Fig. 1c). However, Cu2? and Zn2? exhibit weaker inhibitory effect. Cu2? and Zn2? ions can coordinate with the residues of OxOx as Fe3? does, but they generally have a lower coordination number than that of Fe3?, which may not be able to alter effectively the conformation of OxOx, thus has less effect on the enzyme activity. In fact, they also have different radius. Ca2? ion could not chelate the residues as transition metal ions, thus is unable to inhibit the activity of enzyme. These results clearly explained the controversy results of metal ions inhibition to OxOx in the literature (Sugiura et al. 1979).
Conclusions Interaction of Fe3? ions with OxOx was systematically investigated for the purpose of illustrating the substrate binding site. We found that Fe3? ions are non-competitive inhibitors, upon binding of Fe3? ions the secondary structure of the OxOx was slightly altered. The binding affinity of Fe3? to OxOx was milder. On the basis of the crystal structure of the recombinant OxOx and the intrinsic chemical property of the Fe3? ions, two residue clusters were assigned as potential candidates for the Fe3? ions binding sites. The inhibitory mechanism by Fe3? was further
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understood using the enzyme-substrate docking model, in which the residues that were involved in Fe3? binding play important roles in the substrate orientation. The effective inhibition of Fe3? to OxOx is critically dependent on its size and intrinsic property, therefore different metal ions exhibit different inhibitory activities. The inhibition of Fe3? to OxOx indicates the substrate oxalic acid binds to OxOx through hydrogen bonding with the two residues Asn85 and Asn75. Acknowledgements This work was supported by the State key laboratory of bioreactor engineering (No. 2060204), NSFC of China (No. 20671034), and 111 Project (B07023).
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Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site Yu Pang . Wanjun Lan . Xuelei Huang . Guanke Zuo . Hui Liu . Jingyan Zhang
Received: 5 September 2014 / Accepted: 18 June 2015 / Published online: 24 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe3? ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe3? is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe3? ions, two residue clusters of OxOx were assigned as potential Fe3? binding sites, the mechanism of the inhibition of Fe3? was delineated. Importantly, the residues that interact with Fe3? ions are involved in the substrate orienting based on
Electronic supplementary material The online version of this article (doi:10.1007/s10534-015-9871-7) contains supplementary material, which is available to authorized users. Y. Pang W. Lan X. Huang G. Zuo H. Liu J. Zhang (&) State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China e-mail: [email protected]; [email protected]
computer docking. Consequently, the interaction of OxOx with Fe3? highlights insight into substrate binding site in OxOx. Keywords Oxalate oxidase Fe3? ion Inhibition Computer modeling Substrate binding site
Introduction Oxalate is a toxic compound for human primarily obtained through a diet of oxalate rich plants. High oxalate concentration can cause hyperoxaluria, kidney stones, and cardiological diseases (Hodgkinson 1977; Williams and Wandzilak 1989). Biological research on oxalate degrading enzymes is thus attracting for the potential applications in the oxalate related diseases. Oxalate oxidase (OxOx, EC 1.2.3.4) is one of typical oxalate degrading enzymes, it catalyzes the oxidative cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide (Woo et al. 1998). It is a hexameric enzyme, each subunit possess a barrel structure and a manganese ion locates inside the barrel (Requena and Bornemann 1999). The manganese ion at the active site is bound by four residues (His88, His90, Glu95, and His137) together with two water molecules forming an octahedral coordination environment (Woo et al. 2000). Oxalate oxidation is occurred at the manganese centers. In spite of the successful structural characterization of OxOx in
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yeast, details of its catalytic mechanism remain unclear. It has long been believed that during the catalytic cycle oxalate anion interacts with the MnII center in monodentate mode, and one dioxygen molecule displaced the two coordinated water molecules. However, recently, Whittake et al. proposed that in the resting state, manganese is in oxidation state MnIII, and substrate binds to OxOx forming an enzyme-oxalate complex based on EPR and redox modification studies (Whittaker et al. 2007). Vicky et al. modified OxOx using the specific chemical modifiers to identify surface amino acids that are essential for enzyme activity. They concluded that one carboxylate and one lysine on the per monomer surface was critical for enzyme activity (Kotsira and Clonis 1998). A recent study on OxOx using the complex of the enzyme and substrate analogue glycolate implied that the manganese ion at the active site serves two functions: to organize the substrates (oxalate and dioxygen) and to reduce transiently dioxygen (Opaleye et al. 2006). Several residues are believed critical in orientating the substrates and reaction intermediates for catalysis. There are apparently several key issues of the OxOx mechanisms that are still not well understood, such as substrate binding site, and binding mode etc. In this work, to delineate the substrate binding site, the interaction of Fe3? ions with OxOx was investigated. Effects of various metal ions on the enzyme catalysis, including Na?, K?, Ca2?, Cu2?, Pb2?, and Fe2?were previously investigated to identify the active site before the crystal structure of OxOx was obtained. Monovalent Na?, K? and divalent Mg2?, Zn2?, Cu2?, and Ni2?ions have no effect on the enzyme activity (Kotsira and Clonis 1998). In contrast, earlier work reported that 1 mM of Pb2? marginally activated OxOx, and Pb2? inhibited the enzyme activity at a low concentration (Sugiura et al. 1979). And some reported that high concentration of Fe2? and Fe3? inhibited OxOx almost completely. Whereas Ca2?, Cu2?, and Pb2? stimulated the activity of OxOx at different extents. The mechanism of inhibition or activation to OxOx by metal ions remains unrevealed. However, the effect of metal ions on the activity of OxOx apparently is closely related to the some steps of the enzyme catalysis. Combining with biological methods, spectroscopic method, and computer modeling, interaction of Fe3? with OxOx is systematically studied. We assigned two groups of the
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residues of OxOx that interact with Fe3?, and are critical in orienting substrate binding to the enzyme, hence indicates the substrate binding site.
Materials and methods Materials The barley seeds were provided by Institute of Agriculture, Shanghai Academy of Agriculture Sciences. CMSepharose Fast Flow and Sephacryl, S-200 resins are products of Amersham Biosciences. Horseradish peroxidase (HRP) was purchased from Yuamju Biotech. CO, Lit. Analytic grade oxalic acid, hydrogen peroxide, phenol red, CuSO4, ZnSO4, FeSO4, Fe(NO3)3, MnCl2, CaCl2 were purchased from the Sinopharm Chemical Reagent CO., Ltd. And prepared freshly before the experiment. Enzyme purification OxOx was purified from 10 days old barley roots using the reported method with modifications (Satyapal and Pundir 1993). Frozen roots were ground with chilled deionized water (1:3) at zero degree. The resulting mixture was filtered by cheese cloth first; the filtrate was centrifuged for 30 min at 4 °C giving a crude extract. Solid ammonium sulphate was added to the crude extract to give a final 80 % saturation, and was maintained at 4 °C overnight. The resulting mixture was centrifuged for 30 min and the supernatant was discarded. The sediment was dissolved in distilled water, and the solution was dialyzed against Milli Q water for 16 h. The dialyzed solution was loaded onto CM-Sepharose column (U 2.6 9 30 cm) pre-equilibrated with 0.01 M,pH 4.5 sodium acetate buffer. The enzyme was eluted with a linear gradient of NaCl (0–0.6 M) in the same buffer. The enzyme fractions were pooled and concentrated to *500 lL. Because OxOx is heat stable (Kotsira and Clonis 1997), the pooled fraction from CM-Sepharose column was heat treated (50 °C for 6 min) and centrifuged to remove precipitated protein. The resulting solution was applied on a Sephadex G-200 column (U 1.5 9 75 cm), which is previously equilibrated with 0.05 M sodium succinate buffer (pH 5.0). The active fractions were pooled and concentrated.
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The purity of the concentrated enzyme was checked by SDS-PAGE and stored at -20 °C. Activity assay OxOx can convert oxalate into hydrogen peroxide and carbon dioxide, and the activity of the enzyme was assayed by monitoring the H2O2 using two colorimetric methods, phenol red-HRP (Zuo et al. 2010) and DMA-MBTH methods (Zuo et al. 2010), and the model compound method that developed by our group (Zuo et al. 2010). For DMA-MBTH method, some metal ions may interfere with the assay (Nakano et al. 1985), therefore in this work, phenol red-HRP method was employed mainly. Generally, the reaction mixture (0.2 mL) contained OxOx (0.1 lM), oxalic acid (2 mM) in 20 mM, pH 3.5 sodium succinate buffer was incubated at 40 °C for 5 min, 0.4 mL of sodium succinate buffer (50 mM, pH 5.0) containing phenol red (0.1 mM) and 6.25 U of HRP were then added to detect the reaction product H2O2. The mixture was then incubated at 30 °C for 15 min, 0.1 mL of 5 M NaOH was added to stop the reaction, and the absorbance of the mixture at 610 nm is proportional to the H2O2 concentration in the reaction system. The amount of H2O2 generated during the reaction was calculated according to the standard curve. The effect of metal ions was measured based on the method of enzyme activity assay including metal ions. OxOx was first incubated with the solution of different metal ions for 10 min at 4 °C to ensure metal ions completely bind to OxOx. Microscale thermophoresis (MST) MST was measured with a Nano Temper Monolith NT.115 system (Germany). The concentration of OxOx was 100 nM. OxOx was labeled by Monolith NTTM protein labeling kit with NT-647. The labeled proteins were added to the capillaries (Nano Temper Technologies GmbH) with different concentrations of Fe3? or Ca2?. The binding process of OxOx and Fe3? is characterized by:
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FB =
¼
½AB ½B
½A þ ½B þ KD
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½A þ ½B þ KD Þ2 4½AB 2½B
FB is described by: Fnorm = (1 - FB)Fnorm,unbound ? (FB)Fnorm,bound; Fnorm,unbound: normalized fluorescence of the unbound OxOx; Fnorm,bound: normalized fluorescence of the bound state (Seidel et al. 2013). Circular dichrosim (CD) and UV–Visible measurements A JASCO J815 spectropolarimeter (Jasco International Co. Ltd., Japan) equipped with a Jasco temperature controller (model PTC-423S) and controlled by a PC was used for all circular dichrosim measurements at 22 °C. Milli Q water was used to avoid the interference of the buffer to the CD spectra. A quartz cell with 1 cm path length was used. Each spectrum was averaged from five successive accumulations at a scan rate of 50 nm/min. UV–Visible absorption spectra of the samples were obtained with a Cary 50 spectrophotometer. Computer modeling and docking The crystal structures of OxOx (PDB code 1FI2), were downloaded from http://www.rcsb.org/pdb/ for computer modeling. The surface potential of OxOx was generated by Ds Visualizer 15 program (Accelrys, Inc.). The amino acid residues that interact potentially with metal ions were screened manually within the negatively charged surface area of the OxOx. The docking of the substrate with OxOx was carried out using OxOx with glycolate complex (PDB code: 2ET1) as a template. The docking was performed at ˚ area. the glycolate binding site with a diameter of 10 A The Glide software (Schrodinger, Inc.) was used on linux workstation for the model building.
Results and discussion
A þ B AB
Purification of OxOx
A Fe3? or Ca2?, B OxOx, AB bound complex of OxOx and Fe3?/Ca2?. The equilibrium dissociation constant KD is obtained by fraction bound (FB):
OxOx was purified to homogeneity from barley roots as described in the experimental section. All the
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procedures for isolation and purification of the enzyme were carried out at 4 °C. The overall purification achieved 580-fold activity with a yield of 20.81 % as summarized in Table S1. The purified OxOx exhibited a single protein band in SDS PAGE as shown in Figure S1. The purified enzyme was stored at -20 °C. Interaction of Fe3?with OxOx The inhibitory effect of metal ions on the activity of OxOx though controversial possibly provides information of substrate binding (Zuo and Zhan 2005). It was reported that many monovalent and divalent metal ions are effective to OxOx activity, we focused primarily on Fe3?. As shown in Fig. 1a, OxOx activity was gradually inhibited with the increase of Fe3? concentration, and was almost completely inhibited with 1 mM of Fe3?, IC50 of Fe3? under the condition is 0.6 mM. To preclude the false inhibition caused by the assay method, the activity of OxOx was measured with different methods complementarily and the results are consisted with each other. However, in the different methods as described in experimental section, the activity of OxOx was evaluated by monitoring catalytic product H2O2 (Mongkolisirikieat and Srisuwan 1987). Since H2O2 can easily react with Fe3? through Fenton reaction (Aplin et al. 2001; Zuo and Zhan 2005), the presence of Fe3? could possibly result in a false inhibition. Control experiments with standard H2O2 samples were therefore performed, and the results clearly indicated the assumption of H2O2 by excess Fe3? in the reaction system was not interfered as displayed in Figure S2. On the other hand, metal
Fig. 1 a Inhibitory effect of Fe3? on the activity of OxOx under different Fe3? and pH. Oxalic acid was 2 mM, and OxOx was 0.010 lM. b Inhibitory ability of Fe3? under different concentrations of substrate. Fe3? concentration was 0.5 mM.
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ions such as Fe3?, Mn2?, Ca2? can react with the native substrate oxalic acid forming metal oxalates to consume some oxalic acid, thus will result in a false low activity of OxOx. However, when excess oxalic acid was used, even with very low concentration of Fe3?, the inhibitory effect was still observed. Thus, the inhibitory effect observed with Fe3? was not due to the substrate lose that caused by the formation of oxalates. Intrinsic OxOx contains manganese ions at the active sites, the replacement of manganese ions also could lead to the loss of activity. To exclude the possibility that Fe3? replace Mn2? at the active site, the activity of OxOx was measured in the presence of metalchelating agent EDTA (3.0 mM) in the reaction system, and the activity is slightly affected, which suggested that manganese ions in OxOx are fairly strong chelated by the amino acids residues at the active site, the replacement of Mn2? by other metal ions is unlikely to occur under the assay condition (Mongkolisirikieat and Srisuwan 1987). The activity of the Fe3? inhibited OxOx can be mostly recovered in the presence of excess of EDTA as shown in Figure S3. Taken these results together, we demonstrate clearly that Fe3? ions can inhibit the activity of OxOx. To further confirm the result, inhibition of Fe3? to OxOx was also assayed with different substrate concentrations. As shown in Fig. 1b, in the presence of Fe3?, the amount of the reaction product H2O2 decreased at the different substrate concentrations. The Lineweaver–Burk plot indicated that the Km was not changed in the presence of Fe3? (Figure S4), indicating that Fe3? ion is a non-competitive inhibitor with Ki of 0.75 mM. This is to be expected, because
c MST analysis of Fe3? binding to OxOx. The assay was carried out in 20 mM sodium succinate buffer with 0.05 % Tween-20, and the concentration of OxOx was 0.1 lM
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Affinity of Fe3? to OxOx To measure the affinity of Fe3? to OxOx, microscale thermophoresis (MST) analysis was performed. MST is based on thermophoresis of protein molecules, that allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The molecule mobility in the temperature gradient is analyzed via fluorescence (Seidel et al. 2013). As shown in Fig. 1c, at pH 3.5, with the titration of Fe3?, the fluorescence of protein was decreased, and the binding of Fe3? was observed, comparing to the experiment with Ca2? under the same condition. However, the binding constant of Fe3? was 2.2 mM, which is relatively high. When pH was increased to 6.0, the binding constant decreased dramatically to 0.49 mM, indicating a stronger binding, which is in consistent with the inhibition assay under the same pH (Fig. 1a). This is definitely not a nonspecific electron static interaction, because control experiment with Ca2? did not show any binding. However, such binding constant is relative high, though similar number was reported for other proteins in the literature (Cassland et al. 2004). Inhibitory mechanism of Fe3? The pH dependence of the binding affinity of Fe3? further confirmed the inhibitory of Fe3? was through interaction with the negatively charged areas on the surface of OxOx as shown in Figure S5. To delineate the mechanism of the inhibitory ability of Fe3?, the effect of Fe3? on the OxOx conformation was first examined with circular dichromism (CD). As shown
4 3+
Fe (0.05mM) OxOX 3+ OxOX+ Fe (0.05mM)
3 2 1 0
-1.0
-1
CD (mdeg)
CD (mdeg)
the substrate oxalic acid is more negatively charged, while Fe3? ions are positively charged, in principle they will not bind at the same site of the enzyme. When the experiment was carried out at pH 6.0, which is higher than pI (4.2) of the OxOx, the inhibitory effect is stronger as showed in Fig. 1a compared with the inhibition measured at pH 3.5 (optimum pH for enzyme catalysis). In the presence of 0.4 mM of Fe3?, only *26 % activity was remained at pH 6.0, while about 48 % activity was maintained at pH 3.5. Apparently, when pH of the reaction buffer is above pI of OxOx, the interaction of Fe3? with OxOx is stronger suggesting that Fe3? ions bind at the negatively charged areas of OxOx.
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-2
-1.5
-2.0
-3 210
220
230
Wavelength (nm)
-4 180
200
220
240
260
280
300
Wavelength (nm)
Fig. 2 CD spectra of the OxOx in the presence of Fe3?. The concentration of Fe3? was 0.05 mM, and OxOx was 0.4 lM
in Fig. 2, there are obvious changes in the CD spectrum of the OxOx in 190–250 nm range upon Fe3? ions binding. The positive band at *195 nm, which is typically for a helix and b sheet, increased, and the negative band at 210 nm decreased, while band at 220 nm increased a little bit. The band at 220 nm shifted 5 nm suggesting that the secondary structure of OxOx was slightly affected by the binding of Fe3? ions. However, there is no change in near UV range 260–300 nm, indicating no changes occurred to the aromatic residues of OxOx upon Fe3? ions binding. CD result provides an evidence that Fe3? ions possibly change the secondary structure of the OxOx, which probably is one of the reasons of its inhibition. Fe3? and substrate binding site On the basis of the intrinsic property of Fe3? ion, its non-competitive inhibition to OxOx under different pH conditions, and the inhibition of Fe3? can not be recovered completely by the presence of excess of EDTA, we thus infer that Fe3? ions most likely interact with OxOx at its negatively charged surface. In fact, there are several large negatively charged domains on the surface of electrostatic potential of the OxOx dimmer and monomer (Figure S5). The most possible targets for Fe3? ions are aspartic acid or glutamic acid resides that exposed on the surface of OxOx. Unfortunately, it is not easy to dock a single metal ion with an enzyme using conventional docking programs, such as Z-DOCK or Swiss DOCK. Hence,
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to support this hypothesis, Fe3? ion was manually docked with the OxOx in the negatively charged surface areas. The criteria for locating Fe3? ions are the spatial distances between Fe3? and oxygen and nitrogen atoms of the surface residues that can potentially form a covalent bond with Fe3? ion. Surfing the negatively charged areas on the surface of OxOx, two potential metal ions binding sites, Glu58 and Asp60 (site I), Asn85 and Gln139 (site II) residues clusters were found. As shown in Fig. 3a, at the site I that located on the wall of the b-barrel of the OxOx, Fe3? is surround by four oxygen atoms from Glu58 and Asp60, and two water molecules nearby (Fig. 3b). Similarly, as shown in Fig. 3c, d, Fe3? can be coordinated by the oxygen and nitrogen atoms from Asn85 and Gln139 (site II) and water molecules nearby. The distances between Fe3? and N, O atoms from the residues at these two sites summarized in Table S2 are similar to the distances that observed in many iron containing enzymes and enzyme model compounds (Woo et al. 1998). Crystal structure of the complex of the recombinant OxOx and substrate analogue glycolate revealed that Asn75, Asn85, and Gln139 might be involved in orienting and stabilizing
of the complex of the enzyme with the substrate or intermediates. Site-directed mutagenesis of the two asparagines in the recombinant OxOx also demonstrated that they are essential for OxOx activity (Opaleye et al. 2006). Interestedly, in the residue clusters we identified (Sites I and II), residues Asn75, Asn85, or Gln139 are all directly or indirectly involved. At the site I, Asn85 binds to water molecule that coordinated to Fe3? (Fig. 3b). Atthesite II, Asn85 was involved in chelating to Fe3? (Fig. 3c). The modeling result illustrated the experimental data that the binding of Fe3? could inhibit the activity of OxOx, and inhibition of Fe3? was via interactions with Glu58 and Asp60, Asn85, and Gln139 residues. However, the above result cannot be further established by site-directed mutagenesis, the attempt to clone OxOx gene and express active OxOx in E. Coli was unsuccessful, probably because of its large size (Cassland et al. 2004). Fortunately, crystal structure of OxOx with glycolate was reported, glycolate was regarded as the substrate analogue (Opaleye et al. 2006). Hence, we docked oxalic acid with OxOx based on the crystal structure of OxOx/ glycolate complex, the binding site of glycolate was
Fig. 3 Two potential Fe3? binding sites on OxOx. a site I (Glu58 and Asp60), c Site II (Asn85 and Gln139). c, d are the ball-stick models of the Fe3? coordination at the Sites I and II,
respectively. The red is oxygen, grey is carbon, and light blue is nitrogen, and yellow is manganese. The crystal structure of OxOx (PDB code 1FI2) was used. (Color figure online)
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Fig. 4 a Docking model of OxOx-oxalic acid complex. Two residues (Asn85 and Asn75) together with H2O orient the substrate oxalic acid and stabilize it through hydrogen bonds. The docking of the substrate with OxOx was carried out using OxOx with glycolate complex (PDB code: 2ET1) as a template.
b Inhibitions of Cu2?, Zn2?, and Ca2? ions to the activity of OxOx. The concentration of all the metal ions is 1 mM. Control is the OxOx alone without any extraneous metal ions. The detailed reaction conditions are described in the experimental section
chosen as a binding pocket for oxalic acid. The docking was carried out by further energy minimization. As shown in Fig. 4a, in the best docking model, oxalic acid locates inside barrel, and is in close proximity to the manganese, hydrogen bonding with two residues Asn85 and Asn75. The bond length of ˚ , Mn–O (H2O) is 2.36 A ˚, Mn–O (oxalic acid) is 2.00 A which are quite similar to those found in manganese complexes (Bacelo and Binning 2006; Mason et al. 2009; Scarrow et al. 1994; Smulevich et al. 1995). The ˚, bond length of O(Asn85)–H(Oxalic acid) is 1.61 A ˚ H(Asn75)–O(Oxalic acid) is 1.97 A, and H(H2O)– ˚ , which are short enough to O(Oxalic acid) is 1.61 A form hydrogen bonds. EPR and redox modification studies had showed that oxalic acid binds to OxOx form an enzyme-oxalate complex (Whittaker et al. 2007). The interactions with Asn85 and Asn75 residues apparently play key role in orienting the oxalic acid to the b-barrel, and close to active site. And more importantly, these residues are also involved in the interaction with Fe3?. When Fe3? binds to OxOx, the interaction of these residues with the oxalic acid was interfered, thus the activity of the OxOx was inhibited. The interaction of OxOx with Fe3? highlights insight into substrate binding site in OxOx. Based on the above results, the Site I that assigned by modeling is apparently not a favorable site comparing to the Site II, that’s probably the reason binding affinity of Fe3? is low (Fig. 1c). Generally, the interaction of metal ions with OxOx is dependent on
the intrinsic properties of the metal ions (Fig. 4b). Under the same concentration, Ca2? ion did show any inhibitory effect, which is consistent with its no binding affinity from MST analysis (Fig. 1c). However, Cu2? and Zn2? exhibit weaker inhibitory effect. Cu2? and Zn2? ions can coordinate with the residues of OxOx as Fe3? does, but they generally have a lower coordination number than that of Fe3?, which may not be able to alter effectively the conformation of OxOx, thus has less effect on the enzyme activity. In fact, they also have different radius. Ca2? ion could not chelate the residues as transition metal ions, thus is unable to inhibit the activity of enzyme. These results clearly explained the controversy results of metal ions inhibition to OxOx in the literature (Sugiura et al. 1979).
Conclusions Interaction of Fe3? ions with OxOx was systematically investigated for the purpose of illustrating the substrate binding site. We found that Fe3? ions are non-competitive inhibitors, upon binding of Fe3? ions the secondary structure of the OxOx was slightly altered. The binding affinity of Fe3? to OxOx was milder. On the basis of the crystal structure of the recombinant OxOx and the intrinsic chemical property of the Fe3? ions, two residue clusters were assigned as potential candidates for the Fe3? ions binding sites. The inhibitory mechanism by Fe3? was further
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understood using the enzyme-substrate docking model, in which the residues that were involved in Fe3? binding play important roles in the substrate orientation. The effective inhibition of Fe3? to OxOx is critically dependent on its size and intrinsic property, therefore different metal ions exhibit different inhibitory activities. The inhibition of Fe3? to OxOx indicates the substrate oxalic acid binds to OxOx through hydrogen bonding with the two residues Asn85 and Asn75. Acknowledgements This work was supported by the State key laboratory of bioreactor engineering (No. 2060204), NSFC of China (No. 20671034), and 111 Project (B07023).
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