Crystal Structures of Dye-Decolorizing Peroxidase with Ascorbic Acid and 2

Home , Catalase, Cytochrome c peroxidase, Haloperoxidase, Peroxidase

FEBS Letters 586 (2012) 4351–4356

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Crystal structures of dye-decolorizing peroxidase with ascorbic acid and 2,6-dimethoxyphenol ⇑ Toru Yoshida a, Hideaki Tsuge b, Toru Hisabori a, Yasushi Sugano a,c, a Chemical Resources Laboratory, Tokyo Institute of Technology, R1-8, 4259 Nagatsuta, Yokohama 226-8503, Japan b Faculty of Life Sciences, Kyoto Sangyo University, Motoyama Kamigamo Kita-ku, Kyoto 603-8555, Japan c Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo112-8681, Japan article info abstract

Article history: The structure of dye-decolorizing peroxidase (DyP)-type peroxidase differs from that of other perox- Received 18 September 2012 idase families, indicating that DyP-type peroxidases have a different reaction mechanism. We have Revised 19 October 2012 determined the crystal structures of DyP with ascorbic acid and 2,6-dimethoxyphenol at 1.5 and Accepted 26 October 2012 1.4 Å, respectively. The common binding site for both substrates was located at the entrance of Available online 15 November 2012 the second cavity leading from the DyP molecular surface to heme. This resulted in a hydrogen bond Edited by Miguel De la Rosa network connection between each substrate and the heme distal side. This network consisted of water molecules occupying the second cavity, heme 6-propionate, Arg329, and Asn313. This network is consistent with the proton transfer pathway from substrate to DyP. Keywords: Dye-decolorizing peroxidase Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Crystal structure Substrate binding site Hydrogen bond network

3þ 4þ þ 1. Introduction resting ! compound I : ðFe ÞþH2O2 !ðFe ¼ OÞ þ H2O compound I ! compound II : ðFe4þ ¼ OÞþ þ AH !ðFe4þ ¼ OÞþA Heme peroxidases are a ubiquitous family of enzymes produced compound II ! resting : ðFe4þ ¼ OÞþAH !ðFe3þÞþA þH O by almost all organisms. These enzymes catalyze the reaction, 2

ROOH þ 2AH ! ROH þ 2A þH2O; The parentheses show the state of heme in the peroxidases. In the first step, the enzymes generate a reactive intermediate ‘com- where ROOH represents peroxide, AH represents the substrate, and pound I’ by reaction with H O . In this reaction, H O is reduced to represents an unpaired electron. Generally, heme peroxidases uti- 2 2 2 2 H O, and the enzyme is two-electron oxidized. The iron atom of lize hydrogen peroxide as ROOH. The peroxidase database ‘Peroxi- 2 heme loses one electron, and protoporphyrin loses another elec- Base’ (http://peroxibase.toulouse.inra.fr/index.php) [1] divides tron. In cytochrome c peroxidase (CcP), however, protein radical heme peroxidases into the following six families by organism, pri- is fomed because of quick electron provision from the neighbor mary structure, and substrate: the non-animal peroxidase, animal tryptophan residue to protoporphyrin [2]. Compound I is subse- peroxidase, haloperoxidase, di-heme cytochrome c peroxidase, cat- quently reduced to compound II by the one-electron oxidation of alase, and DyP-type peroxidase families. Except for the latter family, one substrate. Finally, compound II is reduced to its resting state which was identified recently, the reaction mechanisms of all other by the one-electron oxidation of the second substrate, completing families of heme peroxidases have been well characterized. Regard- the catalytic cycle. During this process, an oxygen atom bound to less of their primary structures, peroxidases in these five families the iron atom of compound II is reduced to a water molecule. These have a very similar mechanism of reaction. Their catalytic cycle reactions indicate the existence of two pathways, a proton transfer consists of 3 steps. pathway from substrate to the oxygen atom bound to iron and an electron transfer pathway from substrate to heme. Therefore, substrate binding is required to form these transfer pathways. Abbreviations: APX, ascorbate peroxidase; CcP, cytochrome c peroxidase; DMP, Peroxidase have three substrate binding sites: heme d and c 2,6-dimethoxyphenol; DyP, dye-decolorizing peroxidase; MOPS, 3-Morpholinopro- panesulfonic acid; PDB, protein data bank; VP, versatile peroxidase edges and an exposed tryptophan residue. Substrate complex ⇑ Corresponding author. Fax: +81 3 5981 3667. structures of d edge have been identified in many peroxidases by E-mail address: [email protected] (Y. Sugano). X-ray crystallography [3]. This site is located on the distal side of

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.10.049 4352 T. Yoshida et al. / FEBS Letters 586 (2012) 4351–4356 the heme, allowing the substrate direct contact with heme and acid (MOPS), pH 7.0. Assays were performed at 30 °C on a V-550 suggesting that electrons and protons can transfer directly from UV–Vis spectrophotometer (Jasco). The assay solution for ascorbic substrate to enzyme [4]. In contrast, only three crystal structures acid consisted of 25 mM citrate buffer, pH 3.2, 0.2 mM H2O2, vari- show binding to the c edge: manganese peroxidase and versatile ous concentrations of substrate, and 10 nM DyP, whereas the assay peroxidase (VP) with Mn2+ and ascorbate peroxidase (APX) with solutions for DMP were the same, except for 0.5 nM DyP. Reactions ascorbic acid [3]. These substrates are not located on the heme dis- were initiated by the addition of DyP and monitored by changes in tal side, but binds directly to the 6-propionate of heme via a hydro- absorbance (290 nm for ascorbic acid and 470 nm for DMP). The gen bond, resulting in electron transfer [5,6]. Moreover, ascorbic absorption coefficient of ascorbic acid in 25 mM citrate buffer, 1 1 acid is connected to the water molecules occupying the heme dis- pH 3.2, containing 0.2 mM H2O2, was 655 M cm . The turnover tal side via a hydrogen bond network, consisting of one arginine number (kcat) for DMP was calculated from the absorption coeffi- and several water molecules, resulting in a proton transfer path- cient e = 49.6 [24]. way [7]. In lignin peroxidase and VP, high redox-potential substrate as veratryl alcohol interacts the exposed tryptophan [8], 2.2. Preparation of substrate complex crystals although it is unclear whether a proton is transferred from this substrate [9]. To date, the binding sites of several substrates have Native DyP crystals, prepared as described [21], were soaked been identified in many peroxidases, except for DyP-type overnight in 0.1 M citric acid, pH 4.0, 0.25 M NaCl, 30% (w/v) peroxidases. PEG8000, and 31.9% (v/v) ascorbic acid saturated water or DMP DyP-type peroxidases is a newly identified family of peroxi- saturated dimethyl sulfoxide (DMSO), and briefly in a cryo-protec- dases [10]. The first member of this family identified was DyP from tive solution containing 0.1 M citric acid, pH 4.0, 0.25 M NaCl, 30% Bjerkandera adusta Dec 1, which was found to decolorize dye (w/v) PEG8000, 25% glycerol, and 6.9% (v/v) ascorbic acid saturated wastewater. DyP differed from other peroxidases in that it could water or DMP saturated DMSO. The crystals were subsequently oxidize anthraquinone compounds [11], had a different primary flash frozen in liquid nitrogen. structure than all other peroxidases [12], and did not have well conserved amino acids around the heme moiety [13]. Several genes 2.3. X-ray data collection and structural refinement homologous to DyP have been identified and classified as a novel heme peroxidase family, DyP-type peroxidase. Enzymes in the X-ray crystallography was performed at a wavelength of 1.0 Å DyP-type peroxidase family can be subdivided into types A, B, C, on a beamline PF-AR NW12A instrument at the Photon Factory and D, based on their primary structures [1]. Primary sequence (Tsukuba, Japan). Subsequent procedures, including processing, (amino acid sequence) identities among the 4 types are at most scaling, and refinement, were performed as described [21]. The 15%, with each type having unique characteristics in its primary structures of the substrates were incorporated into the last cycles structure. Type A has a Tat (twin-arginine translocation) signal, of refinement. Data collection and refinement statistics are sum- and is part of a tripartite operon [14]; part of Type B is in an operon marized in Table S1. The figures were prepared using PyMOL soft- with encapsulin [15]; and Type D has an N-terminal prosequence ware (http://pymol.org/). cleaved off during protein maturation [16]. In contrast, DyP-type peroxidases have a similar tertiary structure [10], crystal struc- 3. Results tures of five DyP-type peroxidase have been deposited in Protein Data Bank (PDB): YcdB (type A) [17], BtDyP (type B) [18], TyrA 3.1. Steady state kinetics (type B) [19], DyPB (type B) [20], and DyP (type D) [21]. All of these molecules have a beta-barrel fold, whereas other peroxidases have Although DMP is a substrate of DyP-type peroxidases [16,25– an alpha helix rich structure. In addition, the unique amino acids 27], it is unclear whether ascorbic acid is a substrate. Moreover, on the heme distal side, aspartic acid, arginine, leucine, and phen- in both cases, the kinetic parameter of these substrates have not ylalanine, which differ from those in other peroxidases, are con- been reported. We analyzed these reactions at steady state served in DyP-type peroxidases [21]. (Fig. 1). The oxidation of both substrates fitted the Michaelis–Men- Although the crystal structures of some DyP-type peroxidases ten equation, though the oxidation of DMP showed substrate inhi- have been determined, their reaction mechanism remains unclear. bition in high substrate concentration. The Km for these reaction We previously reported that the aspartic acid residue on the distal were 2.1 mM for ascorbic acid and 2.5 mM for DMP. These values side of the heme moiety was essential for the reaction of this en- are much higher than the Km for Reactive Blue 5 (54 lM) [11]. 1 1 zyme with H2O2 [13]. More recently, however, an arginine residue The kcat were 970 s for ascorbic acid and 1390 s for DMP. located on the distal side of heme, and not this aspartic acid residue, was found to be essential for DyPB peroxidase activity [20], 3.2. Substrate binding site suggesting that the amino acid essential for the reaction with

H2O2 differs according to DyP type. No information, however, is Substrate complex crystals were generated by soaking native available on substrate binding site(s) of DyP-type peroxidase. We crystals of enzyme in reservoirs containing ascorbic acid or DMP. therefore assessed the crystal structures of DyP bound to two sub- Although they showed high Km (low afﬁnity), ascorbic acid and strates, ascorbic acid and 2,6-dimethoxyphenol (DMP). DMP were soluble in water and DMSO, respectively. Therefore, we could prepare soaking solutions with high substrate concentra- 2. Materials and methods tion. This might help success of substrate complex crystals. We found that one ascorbic acid molecule and two DMP molecules

2.1. Steady state kinetics bound to DyP (Fig. 2A), as also shown by Fo–Fc omit electron den- sity maps (Fig. S1). One of the two DMP (DMP1) bound to the same

Substrate and H2O2 solution were freshly prepared in ultrapure site as ascorbic acid, with both ascorbic acid and DMP1 fitting into water. H2O2 concentration was determined using the published a small pocket on the molecular surface of DyP. This site was much 1 1 absorption coefficient (e240 = 39.4 M cm ) [22]. DyP was pre- closer to the heme moiety than the binding site of the second DMP pared with a modification of published procedure [16] and the con- molecule (DMP2) (Fig. 2B). Both ascorbic acid and DMP1 formed centration was determined by the pyridine hemochrome method hydrogen bonds with N313 and H326 (Fig. 2C). No other amino [23]. DyP was diluted with 25 mM 3-Morpholinopropanesulfonic acid residues formed hydrogen bonds with either substrate. Sub- T. Yoshida et al. / FEBS Letters 586 (2012) 4351–4356 4353

tween 6-propionate and each substrate. Similarly, in the APX– ascorbic acid complex, the latter binds directly to 6-propionate by hydrogen bonds, and the propionate is essential for electron transfer [5]. These findings suggest that the DyP substrates transfer electrons to the heme propionate. Moreover, ascorbic acid and DMP1 were connected to the water molecule on the heme distal side of DyP (shown as ‘W1’ in Fig. 2D) through three hydrogen bond networks. Similarly, in the APX–ascorbic acid complex, the latter forms hydrogen bond networks with this water molecule, with this network thought to be the proton transfer pathway [7]. These findings suggest that the hydrogen bond networks between the substrates and the water molecule on the heme distal side of DyP act as proton transfer pathways. A proton donor of ascorbic acid is the C2 hydroxyl group. In DyP–ascorbic acid complex, the C2 hydroxyl group was connected to the heme distal side though the route 3 hydrogen bond network (Fig. 2D). Therefore, the route 3 is likely to be used as the proton transfer pathway in the reaction of DyP with ascorbic acid. A proton donor of DMP is also the hydroxyl group. In DyP–DMP complex, however, the hydroxyl group was connected to the heme distal side though the route 1 and 2. There- fore, the route 1 or 2 is likely to be used as the proton transfer pathway in the reaction of DyP with DMP. We have also compared the structure of DyP with those of other DyP-type peroxidases. In DyP, the heme propionate is buried deep Fig. 1. Steady state kinetics of the reactions of ascorbic acid and DMP with DyP. The within the tertiary structure but is connected to the molecular sur- results of three independent experiments were averaged. (top) Data for ascorbic acid was fitted to the Michaelis–Menten equation. The data of the higher substrate face through the hydrogen bond network of channel 2. The heme concentration could not be measured because of the absorbance unit more than 1. 6-propionate in other DyP-type peroxidases is exposed to the (bottom) Data for DMP showed substrate inhibition at higher DMP concentration, molecular surface (Fig. 3), also allowing the substrate to be con- but the data lower than 3 mM were fitted Michaelis–Menten equation (see inset nected to the heme 6-propionate. This suggests that heme propio- figure). nate is essential for reaction of DyP-type peroxidase. We have also investigated the cavity that connects the molecular surface of DyP to heme. We have previously described a large strate binding did not cause conformational changes in the sur- cavity [21]. Although hydrogen peroxide accesses the heme moiety rounding amino acids. The C1 carbonyl oxygen and the C2 hydro- through a cavity, this cavity was too narrow near the heme to allow xyl group in ascorbic acid and the hydroxyl group and the substrate binding. Here we show that another cavity is present methoxy oxygen in DMP1 replaced two of the water molecules from the molecular surface of DyP to the heme. Since both of these in native DyP (Fig. 2D). Importantly, ascorbic acid and DMP1 were cavities were occupied by water molecules, we have described located close to the 6-propionate and connected to the water mol- these cavities as ‘channels’ that allow solvent access, with channel ecule occupying the heme distal side (W1) through three hydrogen 1 being the cavity previously described, and channel 2 the cavity bond networks: route 1, from substrate through W6, W5, W4, W3, described here (Fig. 4A). Channel 2 has also been observed in DyPB W2, and Arg329 to W1; route 2, from substrate through Asn313, 6- [28]. Interestingly, the water molecules occupying channel 2 were propionate W3, W2, and Arg329 to W1; route 3, from substrate those involved in the hydrogen bond networks from ascorbic acid through Asn313, W9, W8, W7, W3, W2, and Arg329 to W1. This or DMP1 to heme (Fig. 4B). No other channels connected the heme hydrogen bond networks resembled the proton transfer pathway moiety with the molecular surface of DyP. Thus, except for these from ascorbic acid to APX (Fig. 2E) [7]. In contrast, DMP2 formed two channels, there is no binding site on DyP involved in the elec- a hydrogen bond only with the main chain of Ser383 (Fig. 2F). tron and proton transfer pathways from the substrate to the There was no hydrogen bond network from DMP2 to the heme enzyme. moiety. Recently, crystal structure of CcP with two phenol molecules was revealed [29]. This is the only report of peroxidase bound to 4. Discussion a neutral phenol. It was, however, concluded that these bindings were non-specific by inhibition study. Indeed, one phenol does 4.1. Evaluation of the substrate binding site of DyP not have the hydrogen bond network to the heme. On the other hand, another phenol is connected to the heme by a hydrogen bond We found that one ascorbic acid molecule or two DMPs bound network consisting of some water molecules and the main chain of to DyP. However, the DMP2 binding site was far from the heme Ile40 and Gly41. This hydrogen bond network is similar but not moiety, and no hydrogen bond network connected DMP2 to heme. identical to the hydrogen bond networks shown here by us. The These findings suggest that electrons and protons cannot transfer hydrogen bond networks of DyP don’t include a main chain. from DMP2 to DyP. Therefore, we conclude that DMP2 bound to Although it is unclear if this difference is critical for the success DyP non-specifically. In contrast, the ascorbic acid and DMP1 mol- of electron and proton transfer, we believe that the bindings of ecules are connected to heme by hydrogen bond networks, sug- ascorbic acid and DMP1 to DyP are specific for the reasons in the gesting that each of these substrates were bound specifically to previous three paragraphs. DyP. When we compared DyP–substrate complexes (Fig. 2D) and 4.2. Perspective APX–ascorbic acid complex (Fig. 2E), we found that, in the former, ascorbic acid and DMP1 were connected with the 6-propionate by We have shown the binding site on DyP for ascorbic acid and hydrogen bonds through Asn313, and a short distance of 5.4 Å be- DMP. Since the structure of DyP-type peroxidase differed from 4354 T. Yoshida et al. / FEBS Letters 586 (2012) 4351–4356

Fig. 2. Binding sites on DyP for ascorbic acid and DMP. (A) Overall structures of native DyP (left), DyP–ascorbic acid complex (middle), and DyP–DMP complex (right). Native DyP structure is published data: PDB ID 3AFV. Three structures are shown in the view from the same orientation. The molecular surface is shown in white. In native DyP, the translucent molecular surface indicates the location of heme buried within the molecule. Ascorbic acid and DMPs are shown as spheres. (B) Distances between the iron atom of heme and DMPs in DyP–DMP complex. Dotted lines connect the iron atom of heme and the oxygen atom of the DMP hydroxyl group. (C) Magnified views of the common binding site for ascorbic acid and DMP1: (left) native DyP, (middle) DyP–ascorbic acid complex, (right) DyP–DMP complex. The dashed lines represent hydrogen bonds. (D) Hydrogen bond networks connecting the ascorbic acid and DMP1 with the water molecule occupying the heme distal side (W1): (left) native DyP, (middle) DyP–ascorbic acid complex, (right) DyP–DMP complex. Three hydrogen bond networks (three routes) are highlighted: route 1, magenta; route 2, orange; route 3, cyan. The common hydrogen bond network to the three routes is highlighted in gray. The cyan spheres represent water molecules and the red and black circles show the corresponding locations in native DyP and DyP–substrate complexes. The positions of C1 carbonyl, C2 hydroxyl, and C3 hydroxyl in ascorbic acid and 6-propionate in the heme are indicated by the black plain style numbers. (E) Proposed proton transfer pathway from ascorbic acid to heme in APX [7]. Dotted lines represent hydrogen bonds. Red arrows represent the proposed proton transfer pathway. This figure was prepared using PDB ID 1OAF. (F) Magnified view of the DMP2 binding site on DyP–DMP complex.

Fig. 3. Comparison of DyP with three other DyP-type peroxidases: DyP (white), YcdB (yellow), TyrA (magenta), and DyPB (blue). (A) Superimposed structures of four DyP- type peroxidases. (B) Overall structures when the superimposed structures are viewed from the same orientation. The black square in the DyP represents the location of channel 2. The magniﬁed views of the upper black squares are shown at the bottom. T. Yoshida et al. / FEBS Letters 586 (2012) 4351–4356 4355

Fig. 4. Location of the two cavities from the molecular surface to the heme moiety in DyP. Two cavities are represented by the name of channel 1 and channel 2, respectively. (A) Overall structure of native DyP. The molecular surface is shown in translucent white. The blue and cyan spheres represent water molecules occupying channels 1 and 2, respectively. (B) Cutaway view showing the shapes of channels 1 and 2. Channel 1 leads from the molecular surface to the heme iron atom. Channel 2, in the shape of a doughnut, leads from the molecular surface to Arg329. Dotted lines represent hydrogen bonds.

those of other peroxidases, we suspected that the reaction mecha- enzymatic use of the Grotthuss mechanism. J. Am. Chem. Soc. 133, 15376– nisms would also differ. Surprisingly, however, the DyP binding 15383. [8] Doyle, W.A., Blodig, W., Veitch, N.C., Piontek, K. and Smith, A.T. (1998) Two site was similar to that on APX for ascorbic acid, suggesting that substrate interaction sites in lignin peroxidase revealed by site-directed reactions always take place near the heme propionate. In contrast, mutagenesis. Biochemistry 37, 15097–15105. no information is yet available on the DyP binding sites for anthra- [9] Wariishi, H., Huang, J., Dunford, H.B. and Gold, M.H. (1991) Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic quinone compounds, which are unique substrates of DyP-type per- characterization. J. Biol. Chem. 266, 20694–20699. oxidases. Soaking native crystals of enzyme in solutions of these [10] Sugano, Y. (2009) DyP-type peroxidases comprise a novel heme peroxidase family. Cell. Mol. Life Sci. 66, 1387–1403. compounds was unsuccessful, despite their much lower Kms com- [11] Kim, S.J. and Shoda, M. (1999) Purification and characterization of a novel pared to those of ascorbic acid and DMP. This may be due to the peroxidase from Geotrichum candidum Dec 1 involved in decolorization of low solubility of these compounds and/or to crystal packing, allow- dyes. Appl. Environ. Microbiol. 65, 1029–1035. ing no space for the binding of bulky anthraquinone compounds. [12] Sugano, Y., Sasaki, K. and Shoda, M. (1999) CDNA cloning and genetic analysis of a novel decolorizing enzyme, peroxidase gene dyp from Geotrichum The elucidation of this binding site will shed light on the differ- candidum Dec 1. J. Biosci. Bioeng. 87, 411–417. ences in reaction mechanisms of DyP-type and other peroxidases. [13] Sugano, Y., Muramatsu, R., Ichiyanagi, A., Sato, T. and Shoda, M. (2007) DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase Acknowledgments family: Asp171 replaces the distal histidine of classical peroxidases. J. Biol. Chem. 282, 36652–36658. [14] Sturm, A., Schierhorn, A., Lindenstrauss, U., Lilie, H. and Brüser, T. (2006) YcdB This research was undertaken with the assistance of the Photon from Escherichia coli reveals a novel class of Tat-dependently translocated Factory in KEK (proposal numbers 2010G011 and 2012G076) and hemoproteins. J. Biol. Chem. 281, 13972–13978. [15] Sutter, M., Boehringer, D., Gutmann, S., Günther, S., Prangishvili, D., Loessner, was supported by a Grant-in Aid for Scientific Research (No. M.J., Stetter, K.O., Weber-Ban, E. and Ban, N. (2008) Structural basis of enzyme 22570136) from the Japan Society for the Promotion of Sciences. encapsulation into a bacterial nanocompartment. Nat. Struct. Mol. Biol 15, 939–947. [16] Sugano, Y., Nakano, R., Sasaki, K. and Shoda, M. 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