APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2009, p. 7509–7518 Vol. 75, No. 230099-2240/09/$12.00 doi:10.1128/AEM.01121-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of a Novel Peroxidase from theCyanobacterium Anabaena sp. Strain PCC 7120�

Henry Joseph Oduor Ogola,1 Takaaki Kamiike,1 Naoya Hashimoto,1 Hiroyuki Ashida,2Takahiro Ishikawa,1 Hitoshi Shibata,1 and Yoshihiro Sawa1*

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University,1 andDepartment of Molecular and Functional Genomics, Center for Integrated Research in Science, Faculty of Life and

Environmental Science, Shimane University,2 Matusue, Shimane 690-8504, Japan

Received 15 May 2009/Accepted 26 September 2009

The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase(Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We clonedand heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperatureof 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with abroad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzymeefficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, ReactiveBlue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256�M � min�1, respectively. The apparent Km and kcat/Km values for Reactive Blue 5 were 3.6 �M and 1.2 � 107

M�1 s�1, respectively, while the apparent Km and kcat/Km values for H2O2 were 5.8 �M and 6.6 � 106 M�1 s�1,respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but wassignificantly enhanced 2- to �50-fold in the presence of the natural redox mediator syringaldehyde. Thespecificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application ofAnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this studyrepresents the only extensive report in which a bacterial DyP has been tested in the biotransformation ofsynthetic dyes.

In textile, food, and dyestuff industries, reactive dyes such asazo and anthraquinone (AQ) and pthalocyanine-based dyesconstitute one of the extensively used classes of synthetic dyes.However, it has been estimated that approximately 50% of theapplied reactive dye is wasted because of hydrolysis during thedyeing process (26, 35). This results in a great effluent problemfor the industries because of the recalcitrant nature of thesedyes. With increased public concern and ecological awareness,in addition to stricter legislative control of wastewater dis-charge in recent years, there is an increased interest in variousmethods of dye decolorization. Dye decolorization using phys-icochemical processes such as coagulation, adsorption, andoxidation with ozone has proved to be effective. However,these processes are usually expensive, generate large volumesof sludge, and require the addition of environmentally hazard-ous chemical additives (26). There are several reports of mi-croorganisms capable of decolorizing synthetic dyes. This hasbeen attributed to their growth and production of enzymessuch as laccase (1, 9, 40), azoreductases (3), and peroxidases,for example, lignin peroxidase (12, 25, 36), manganese perox-idase (10, 38), and versatile peroxidase (16). However, most ofthe synthetic dyes are xenobiotic compounds that are poorly

degraded using the typical biological aerobic treatments. Fur-thermore, microbial anaerobic reductions of synthetic dyes areknown to generate compounds such as aromatic amines thatare generally more toxic than the dyes themselves (3). There-fore, for environmental safety, the use of enzymes instead ofenzyme-producing microorganisms presents several advan-tages such as increased enzyme production, enhanced stabilityand/or activity, and lower costs by using recombinant DNAtechnology.

Peroxidases are heme-containing enzymes that use hydrogenperoxide (H2O2) as the electron acceptor to catalyze numerousoxidative reactions. They are found widely in nature, both inprokaryotes and eukaryotes, and are largely grouped into plantand animal superfamilies. They are one of the most studiedenzymes because of their inherent spectroscopic propertiesand potential use in both diagnostic and bioindustrial applica-tions. In particular, their ability to degrade a wide range ofsubstrates has recently stimulated interest in their potentialapplication in environmental bioremediation of recalcitrantand xenobiotic wastes (10, 25, 26).

Recently, a novel family of heme peroxidases characterizedby broad dye decolorization activity has been identified invarious fungal species such as Thanatephorus cucumeris Dec1(18), Termitomyces albuminosus (15), Polyporaceae sp. (15),Pleurotus ostreatus (13), and Marasmius scorodonius (27). Be-cause of their broad substrate specificity, low pH optima, lackof a conserved active site distal histidine, and structural diver-gence from classical plant and animal peroxidases (32), theseproteins have been proposed to belong to the novel DyP per-

* Corresponding author. Mailing address: Department of Life Sci-ence and Biotechnology, Faculty of Life and Environmental Science,Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504,Japan. Phone: 81 852 32 6586. Fax: 81 852 32 6585. E-mail: ysawa@life.shimane-u.ac.jp.

� Published ahead of print on 2 October 2009.

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oxidase family. Over 400 proteins of prokaryotic and eukary-otic origins have been grouped in the DyP peroxidase family,Pfam 04261 (http://pfam.sanger.ac.uk/), and it is apparent fromgenome databases that many species possess DyP. The abilityof these proteins to effectively degrade hydroxyl-free AQ andazo dyes as well as the specificity for typical peroxidase sub-strates illustrates their potential use in the bioremediation ofwastewater contaminated with synthetic dyes. However, withthe exception of a DyP from the plant pathogenic fungus T.cucumeris Dec1 (an anamorph of Rhizoctonia solani, a verycommon fungal plant pathogen), which has been characterizedextensively (18, 28, 30–32, 34), little information is available onother members of the DyP family. In particular, studies onbacterial DyPs have been limited to only the automaticallytranslated sequence or structural data (41, 42). Within thecontext of further understanding the structure-function andpotential applicability of these novel types of enzymes in gen-eral, we have taken an interest in DyP-type enzymes, particu-larly, the less known bacterial groups.

Cyanobacteria (blue-green algae) represent the most prim-itive, oxygenic, plant-type photosynthetic organisms and arethought to be involved in greater than 20 to 30% of the globalphotosynthetic primary production of biomass, accompaniedby the cycling of oxygen. Anabaena sp. strain PCC 7120 is afilamentous, heterocyst-forming cyanobacterium capable of ni-trogen fixation and has long been used as a model organism tostudy the prokaryotic genetics and physiology of cellular dif-ferentiation, pattern formation, and nitrogen fixation (14).This strain’s genome sequence is complete and annotated (17).From bioinformatics analysis of the Anabaena sp. strain PCC7120 genome, we identified an open reading frame (ORF),alr1585, encoding a putative heme-dependent peroxidase ex-hibiting homology to T. cucumeris Dec1, DyP. Here, we reporton the characterization of this novel bacterial DyP, designatedAnaPX (for Anabaena peroxidase), from the cyanobacteriumAnabaena sp. strain PCC 7120, with broad specificity for botharomatic compounds and synthetic dyes such as AQ dyes.

MATERIALS AND METHODS

Bacterial strains and materials. The Anabaena sp. strain PCC 7120 (ATCC27893) was obtained from the ATCC (Virginia). Cell cultures were grown inBG11 medium at 30°C in air under continuous illumination (40 �E m�2 s� 1).E. coli strains MV1184 [ara �(lac proAB) rpsL thi (�80 lacZ�M15) �(srlrecA)306::Tn10 (Tetr) F� [traD36 proAB� lacIq lacZ�M15]] and DH5�[� �80dlacZ�M15 �(lacZYA-argF)U196 recA1 endA1 hsdR17(rK

� mK�)

supE44 thi-1 gyrA relA1] were used as host bacteria for standard recombinantconstructions while katGE-disrupted E. coli BL21(DE3) [F� ompT hsdSB(rB

�

mB�) thi-1 hfrH katE12::Tn10 supE44 hsdR endA1 pro thi katG::Tn5 gal dcm

(DE3)] (kat mutant E. coli) was used for overexpression of AnaPX. Reactive Red33, Reactive Yellow 2, and Direct Yellow 12 were kind gifts from Nippon KayakuLtd. (Tokyo, Japan). Other reactive dyes were obtained from Sigma-AldrichJapan (Tokyo, Japan), ICN Biomedicals (Ohio), DyStar Japan (Tokyo, Japan),and Waldeck-Gmbh & Co. (Munster, Germany) were used without furtherpurification. Horseradish peroxidase ([HRP] Reinheit Zahl value of 3.0; 99%purity) in lyophilized form, purified from horseradish roots, was purchased fromOriental Yeast Co. Ltd. (Tokyo, Japan), and used without further purification.All other reagents and chemicals used were commercially available and of re-agent grade.

Cloning and overexpression of recombinant AnaPX. Genomic DNA was pre-pared from Anabaena sp. strain PCC 7120 cells according to Ausubel et al.(2). The ORF alr1585 (http://genome.kazusa.or.jp/cyanobase/Anabaena/genes/alr1585) was amplified by the following two oligonucleotide primers: a senseprimer (5�-GGAATTCGtaaAGGATTAATTCATGGCAC-3�) containing anEcoRI recognition site (in boldface), a stop codon (lowercase letters), and a

Shine-Dalgarno sequence (underlined) located 7 nucleotides upstream of thestart codon ATG (in boldface and underlined) of AnaPX; and an antisenseprimer (5�-CAGCTGCAGGCATAAAAATGTTCTC-3�) containing an PstI rec-ognition site (in boldface). PCR consisted of initial denaturation at 95°C for 1min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°Cfor 1 min, and extension at 72°C for 2 min, with a final extension step at 72°C for5 min. The amplified DNA was subcloned into pT7 Blue T-vector (Novagen,WI), and DNA sequences of both strands were checked by an ABI Prism 3100Genetic Analyzer (Applied Biosystems, CA) using a BigDye Terminator, version3.1, Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The insertedDNA was digested with EcoRI and PstI and then ligated into the respective sitesof pUC18 vector (Takara Bio Ltd., Otsu, Japan). The resultant plasmid wasdesignated pUC-AnaPX; in this construction, AnaPX was under the control ofthe lac promoter. A kat mutant E. coli BL21(DE3) strain (with disrupted catalasegenes [�katE �katG]) was transformed with pUC-AnaPX, and the recombinantcells were used for the overproduction and purification of Anabaena sp. strainPCC 7120 peroxidase.

The transformed cells were incubated with reciprocal shaking at 37°C in LBmedium containing 50 �g/ml ampicillin. After overnight cultivation, the culturewas inoculated into the same medium, supplemented with 1 mM 5-aminolevulincacid and 10 �M hemin chloride, followed by incubation with shaking at 37°Cuntil an optical density at 600 nm of �0.8 was reached. Isopropyl-�-D-thiogalac-topyranoside was then added to a final concentration of 0.1 mM to induce the lacpromoter, and further cultivation was carried out at 25°C for 18 h. Cells wereharvested by centrifugation (10,000 � g) and washed twice with 50 mM potas-sium phosphate (KP) buffer, pH 7.0, and the pellet was stored at �80°C untilpurification.

Purification of recombinant AnaPX. The harvested cells were suspended in 50mM KP buffer, pH 7.0 (buffer A), disrupted by a French press, and cell debriswas removed by ultracentrifugation. The resulting supernatant was dialyzedovernight against 5 liters of buffer A. The dialyzed lysate was loaded onto aToyopearl DEAE-650M column (2.5 by 20 cm; Tosoh Corp., Tokyo, Japan)equilibrated with buffer A and eluted with a linear gradient of 0 to 500 mM NaCl.The active fractions were pooled, solid ammonium sulfate was added to a 20%saturated concentration; fractions were subjected to a Toyopearl Butyl-650Mcolumn (2.5 by 20 cm; Tosoh Corp.) equilibrated with buffer A containing 20%saturated ammonium sulfate and eluted with a linear gradient of 20 to 0%saturated ammonium sulfate. The active fractions were pooled and desalted bya PD 10 column (Amersham Pharmacia Biotech, NJ). The resultant fraction wassubjected to a Hypatite C (hydroxylapatite) column (2 by 15 cm; ClarksonChemical, PA) equilibrated with 10 mM KP buffer, pH 7.0, and washed with 5volumes of buffer A, and the protein was eluted with a linear gradient of 0 to 200mM KP. The active fractions containing purified enzyme were pooled and de-salted by a PD 10 column. The homogeneity of the purified protein was con-firmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Enzymatic analysis. AnaPX activity was assayed with a spectrophotometer(UV-1700; Shimadzu, Kyoto, Japan) at the maximum absorption wavelength ofeach dye and model compound at pH 4.0. Measurement of enzyme activity wasinitiated by the addition of 0.4 mM H2O2 at 37°C except for the assay of optimaltemperature for decolorization. One unit of enzyme activity was defined as theamount of enzyme required for the decolorization of 1 �mol of Reactive Blue 5(RB5) per min in the reaction mixtures. For dye decolorization experiments, thereaction mixtures contained 1 absorbance unit of dye at the maximal visibleabsorbance wavelength and 2.1 nM enzyme in 50 mM citrate buffer (pH 4.0 to4.4), and the reactions were initiated by 0.4 mM hydrogen peroxide addition orin the presence of 40 �M syringaldehyde for mediator experiments. Also, eachdye was treated under similar conditions for 2 h, and the decrease in absorbanceof the dye solutions at their respective maximum wavelength (max) was moni-tored. The percent decolorization was calculated by taking untreated dye solu-tion as a control (100%). The wavelengths and absorption coefficients used forvarious substrates were as follows: guaiacol, ε470 26.6 mM�1 cm�1; pyrogallol,ε430 2.47 mM�1 cm�1; ascorbate, ε290 2.8 mM�1 cm�1; 4-aminoantipyrine,ε510 6.58 mM�1 cm�1; D-iso-ascorbate, ε290 3.3 mM�1 cm�1; syringalde-hyde, ε320 8.5 mM�1 cm�1; ABTS [2,2�-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid], ε436 29.3 mM�1 cm�1; NADH and NADPH, ε340 6.2mM�1 cm�1.

The thermostability of AnaPX was determined by monitoring the change inperoxidase activity at different temperatures (30°C, 40°C, 50°C, and 60°C) in 50mM KP buffer, pH 7.2. The residual activity was determined at appropriateintervals using a standard assay method. To determine its pH stability, theenzyme was incubated for 30 min at 37°C at various pHs, and the remainingactivity was again determined using a standard assay. Kinetic parameters of

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AnaPX activities for H2O2 and RB5 were calculated by decolorization of the dyeat 600 nm (ε 11 mM�1 cm�1).

Molecular mass, polyacrylamide gel electrophoresis, and protein determina-tion. The purified enzyme sample was applied to a Superdex 200HR 10/30column, which was attached to a BioLogic high-performance liquid chromato-graph (Bio-Rad Japan, Tokyo, Japan), and then eluted with 50 mM KP buffercontaining 0.15 M NaCl at a flow rate of 0.5 ml/min. The absorbance of theeffluent was recorded at 280 nm. The molecular mass of the enzyme was calcu-lated from the mobilities of the standard proteins glutamate dehydrogenase (290kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32kDa), and cytochrome c (12.4 kDa). Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry analyses were also used to determine proteinmolecular weights, and analysis was performed using a Voyager-DE PRO Bio-spectrometry Workstation mass spectrometer (Applied Biosystems, CA). In ad-dition, SDS-PAGE was performed according to Laemmli (19) using a 12%polyacrylamide gel and gels stained with Coomassie brilliant blue R250. Isoelec-tric focusing was performed by a PhastSystem (GE Japan, Tokyo, Japan), usinga Phastgel IEF 3-9 (GE Japan) and a broad pI calibration kit (pH 3 to 10). Allprotein contents were determined by a Bradford dye-binding assay using bovineserum albumin as a standard.

NH2-terminal amino acid sequencing. The sequence of the first 8 amino acidsat the N terminus of the purified AnaPX was determined by stepwise Edmandegradation in a pulse-liquid automated sequencer (Model PPSQ-10; Shimadzu,Kyoto, Japan) coupled with high-performance liquid chromatography. One mi-crogram of homogenously purified enzyme was applied to glass wool and thenused directly in the sequencer.

Determination of the prosthetic group. Heme was identified by UV absorptionspectroscopy, and its stoichiometry was estimated by conversion to the pyridinehemochrome (5). This procedure consisted of adding pyridine (final concentra-tion, 20%) and then KOH (final, 50 mM) and a small amount of Na2S2O4 to theenzyme. After 15 min, the spectrum of the pyridine hemochrome was recorded.The hemochrome was determined using the published molar absorption coeffi-cient (5) while the Soret band molar absorption coefficient at 404 nm (ε404) wasdetermined on a dry-weight basis. Enzyme (1.9 �M) was dissolved in 50 mM KPbuffer (pH 7.0), and the CN-adducts were obtained by addition of 1 mM KCN forspectral analysis. The oxidized AnaPX (5 ��) was scanned after of addition ofequimolar H2O2 to enzyme in 50 mM citrate buffer (pH 4.4). The absorptionspectra of all samples were measured by a Beckman DU-7400 spectrophotom-eter (Beckman Coulter Inc., CA) at 25°C.

Homology modeling of AnaPX and docking simulation of a possible substratebinding pocket. To gain understanding of the differences in substrate specificitiesbetween AnaPX and fungal DyP, homology modeling using the T. cucumerisDec1 DyP peroxidase crystal structure (PDB code 2d3q) as a template wasperformed using the Molecular Operating Environment (MOE) homology pro-gram, MOE 2008.1001 (Chemical Computing Group, Montreal, QC, Canada),based on a segment-matching procedure (20) and a best-intermediate algorithmwith the option to refine each individual structure enabled. A database of 10structures that were each individually refined to a root mean square gradient of1 Å was generated. The stereochemical quality of the refined model was assessedby using Ramachandran plot analysis, and structural analysis was performedusing the Protein Geometry function of the MOE Protein Structure Evaluationprogram, which searches for disallowed bond angles, bond lengths, and sidechain rotamers. To obtain a final model that had a few outlier residues, afine-grain energy minimization and a molecular dynamics simulation were per-formed in the models. The obtained final model was further validated withVERIFY3D (21) available from NIH MBI Laboratory Servers. The three-di-mensional (3D) profile score of the final model was 131 (Scalc), while the pre-dicted 3D profile score for a 469-residue protein was 215. Since the ratio of thecalculated 3D profile score to the 3D predicted profile score was 0.61 (0.45),the final model was evaluated as an adequate model.

Docking simulation was performed for substrate (guaiacol and RB5) bindingto the AnaPX model and the T. cucumeris Dec1 DyP peroxidase (PDB code2d3q) substrate access channel using the MMFF94x force field distributed in theMOE 2008.1001 program with the Monte Carlo docking procedure of ASE-Dock2005 (Ryoka Systems, Tokyo, Japan). Prior to docking, the energy of the AnaPXmodel or the T. cucumeris Dec1 DyP structure was minimized using the Amber99force field. In the simulation experiment, 32 and 10 solutions were generated fordocking guaiacol and RB5, respectively, in the AnaPX model while in T. cu-cumeris Dec1 DyP, 19 and 6 docking solutions were generated for guaiacol andRB5, respectively. Solutions were ranked according to total interaction energy(i.e., the summation of intermolecular electrostatic, van der Waals, and ligandenergy), and each best-ranked docking solution for guaiacol and RB5 (each

structural member was within 10 kcal/mol from the lowest energy structure) wasconsidered as a candidate substrate-peroxidase complex for the two enzymes.

RESULTS

AnaPX is a member of the novel DyP family. We performeda BLAST search of the T. cucumeris Dec1 DyP sequenceagainst the protein databases and identified alr1585 as an ORFthat encodes a putative heme-dependent peroxidase inAnabaena sp. strain PCC 7120, a photoautotrophic heterocys-tous cyanobacterium, with 28% amino acid homology to the T.cucumeris Dec1 DyP. We also detected homologues in threecyanobacterial species: Anabaena variabilis ATCC 29413, Cya-nothece sp. strain PCC 8801, and Cyanothece sp. strain ATCC51142, with 94%, 38%, and 41% homologies to alr1535, re-spectively. According to the classification system of the perox-idase database (PeroxiBase) of the University of Geneva (http://peroxibase.isb-sib.ch/classes.php), DyPs are classified intofour distinct classes on the basis of their homologies. Classes A,B, and C generally include DyPs of bacteria belonging to thephyla Firmicutes, Actinobacteria, and Alpha-, Beta-, Gamma-,and Deltaproteobacteria, while class D includes fungal DyPs.However, alr1585 and the cyanobacterial DyP homologues ex-hibited considerably lower homologies to other bacterial DyPs(�15%) than to fungal homologs (25%) and were thus pu-tatively grouped together with fungal DyP in class D (Fig. 1b).At the amino acid level, AnaPX showed a conserved heme-binding site, especially the catalytically important distal andproximal residues (Fig. 1a), and the typical distal GXXDGmotif of DyPs. It was possible to predict several catalyticallyimportant residues, except for a proximal aspartate residuethat was replaced with glutamate (Glu391) in T. cucumerisDec1. On the proximal side, His331 should serve as the fifthligand of the heme iron, while Asp419 should be its hydrogen-bonded partner, forming a Fe-His-Asp triad also conserved inthe plant peroxidase superfamily.

Purification of recombinant AnaPX. The alr1585 fromAnabaena sp. strain PCC 7120, which encodes AnaPX, wascloned, sequenced, and expressed at a considerable level inE. coli. Overexpression of pUC-AnaPX using the lac pro-moter system was optimized in a kat mutant E. coliBL21(DE3) strain grown in LB medium supplemented with5-aminolevulinc acid at 25°C. To prevent the formation of aLacZ alpha-AnaPX fusion protein, a stop codon and a newribosomal binding site (the Shine-Dalgarno sequence) wereintroduced 7 nucleotides upstream of the AnaPX startcodon ATG in the sense primer. The expressed recombinantAnaPX was purified from the cell extract to apparent ho-mogeneity in three chromatographic steps, as described inthe Materials and Methods section. Using the above expres-sion system, an appreciable enzyme yield of �10 mg/liter ofculture could be achieved. During purification, trace cata-lase activity observed in the crude enzyme extract was com-pletely lost, indicating that the enzyme lacks catalase activ-ity. Table 1 shows the typical results of the purification ofrecombinant AnaPX. We identified the 8 residues from theN terminus of AnaPX by Edman degradation. The N-termi-nal amino acid sequence of AnaPX (ALTEKDLK) beginswith an alanine residue, indicating the loss of methionineduring protein processing in E. coli. However, the N-termi-

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nal sequence exactly matched the amino acid sequence (res-idues 2 to 8) deduced from the nucleotide sequence. Theestimated molecular masses by SDS-PAGE and matrix-as-sisted laser desorption ionization mass spectrometry analy-sis were �54 kDa (Fig. 2, lane 2) and 53,280 Da, respec-tively. Indeed, these findings agree with the calculated

molecular mass (53,985 Da) of both the deduced amino acidsequence (53,368 Da) and the heme moiety (617 Da). Sizeexclusion chromatography using a Superdex 200HR 10/30column yielded an experimental native molecular mass of209 kDa.

Properties of purified AnaPX. Fig. 3 depicts the stability ofthe enzyme to temperature, pH, and H2O2. The enzyme wasconsiderably more stable at 30°C and 40°C, where it retainedmore than 90% of its activity. In addition, the enzyme retained90% activity when stored in 50 mM KP buffer, pH 7.0, at 4°Cfor 40 days, illustrating the robustness of the enzyme tet-rameric structure under the storage conditions. However, theenzyme lost more than 90% of its activity after incubation at50°C and 60°C for 3 h (Fig. 3a). Evaluation of the enzymeactivity (decolorization of RB5 dye) at different temperaturesranging from 25°C to 45°C revealed an optimum temperatureof approximately 35°C and pH optima of 4.0 to 4.4 (Fig. 3b).

FIG. 1. Sequence alignment and phylogenetic analysis of AnaPX and other related DyP proteins. (a) Multiple sequence alignment of Anabaenasp. strain PCC 7120 AnaPX (accession no. Q8YWM0 [ANASP]), cyanobacterial homologs of A. variabilis ATCC 29413 (Q3M5E1 [ANAVT]),Cyanothece sp. strain PCC 8801 (B1U6L6 [SYNP8]), and a fungal DyP of T. cucumeris Dec1 (Q8WZK8 [TcDec1]) showing the strictly conservedresidues (black shading) and regions of less strict conservation (gray shading). The unique distal motif GXXDG (boxed) of DyP proteins andconserved active-site residues (asterisks) are also depicted. (b) Unrooted neighbor-joining bootstrap tree constructed from alignment of AnaPXwith related DyPs.

TABLE 1. Purification of recombinant AnaPX

Enzyme fraction Activity(U)

Amt ofprotein

(mg)

Specificactivity

(U mg�1)

Purification(n-fold)

Yield(%)

Crude extract 5,420 968 5.6 1 100Toyopearl

DEAE-650M4,932 379 13 2.3 91

ToyopearlButyl-650M

4,282 38 114 19.6 79

Hypatite C 2,601 9 283 50.5 48

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When maintained at 40°C for 20 min, the enzyme was stable atpH values between 3.5 and 9.5 (Fig. 3c). AnaPX also exhibiteda higher tolerance to peroxide than HRP (Fig. 3d), where itretained 50% of its activity at 2.5 mM H2O2 compared to

that observed for HRP at 1 mM H2O2. The apparent optimumconcentration of H2O2 required for the decolorization of RB5by AnaPX was 0.4 mM; thus, this concentration was used forthe peroxidase analysis experiments. Isoelectric focusing withthe PhastSystem yielded only one band with a pI of 3.68 (datanot shown).

Absorption spectra of AnaPX. The purified enzyme wasbrownish red in the solution, showing the existence of theheme group. The UV-visible spectrum of resting-state AnaPXshowed Soret, �, and charge transfer band maxima at 404, 500,and 630 nm, respectively (Fig. 4a). The A404/A280 ratio, or theReinheit Zahl value, which reflects the purity and spectralcharacteristics of hemoproteins, was 1.4, with a molar absorp-tion coefficient of 106 mM�1 cm�1 at 404 nm. As shown in Fig.4a (inset), the pyridine hemochrome of AnaPX had absorptionpeaks at 416 (Soret band), 524 (� band), and 557 nm (� band)that are characteristic of iron protoporphyrin IX. By using amolar absorption coefficient of 34.5 mM�1 cm�1 at 557 nm forpyridine hemochrome, the heme content was estimated to be0.91 mol per mole of protein. The reduction of the enzymewith dithionite decreased the Soret band and shifted it to 435nm, and a new peak appeared at 557 nm, while the addition ofcyanide resulted in a shift of the Soret band to 423 nm and theformation of a new peak at 537 nm as the peaks at 500 and 600nm decreased (Fig. 4a). In the presence of equimolar H2O2 at

FIG. 2. SDS-PAGE of crude extract of E. coli BL21 overexpressingAnaPX (lane 1) and purified recombinant AnaPX (lane 2). The crudeextract (20 �g) and pure enzyme (10 �g) were stained by Coomassiebrilliant blue. The arrow shows the position of the AnaPX band (54kDa). M, molecular mass standard (kDa).

FIG. 3. Thermostability (a), pH optimum (b), pH stability (c), andH2O2 stability (d) of AnaPX. Enzyme activity was measured afterpreincubation for 3 h at various temperatures in 50 mM KP buffer, pH7.0 (a); on 2 mM guaiacol (F and E) and 0.1 mg/ml RB5 (� and ƒ) atdifferent pHs in citrate-citric acid buffer (closed symbols) and KP(open symbols) under standard conditions, with the highest enzymeactivity for each substrate set to 100% (b); after preincubation at 40°Cfor 20 min in 100 mM citrate-citric acid buffer (F), KP (E), andNH4Cl-NH4OH (�) buffers (c); and for AnaPX (F) and HRP (E)after preincubation in various H2O2 concentrations at 37°C for 20 minin 100 mM KP buffer, pH 7.0 (d).

FIG. 4. Absorption spectra of AnaPX. (a) Spectra of the restingstate (thick line) of AnaPX (1.9 �M) dissolved in 50 mM KP buffer, pH7.0. The CN-adduct (dashed line) and reduced form of AnaPX (dottedline) were obtained by addition of 5 mM KCN and a small amount ofsodium dithionite, respectively. The inset shows a pyridine hemochro-mogen spectrum of AnaPX. (b) The spectrum of AnaPX (5 �M) andcompound I species obtained in the presence of equimolar H2O2 in 50mM citrate buffer, pH 4.4. AU, arbitrary units.

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pH 4.4, AnaPX was oxidized to compound I, characterizedwith a shift in the Soret band to 401 nm with additional peaksat 359 (shoulder), 524, 557 (shoulder), and 651 nm (Fig. 4b).

Enzyme activity inhibitors. AnaPX also showed variable in-hibition profiles in the presence of various inhibitor com-pounds. In particular, AnaPX was highly sensitive to the sui-cide substrate phenylhydrazine (1 mM), Fe2� (5 mM), andNaN3 (10 mM), a typical peroxidase inhibitor, with 100%,98.8%, and 82%, respectively. However, it should be notedthat the enzyme was not sensitive to the prototypical catalaseinhibitor 3-amino-1,2,4,-triazole in the presence of ascorbicacid (1.0 mM). Mn2� also had a significant effect on enzymeactivity, with 57.1% inhibition at 5 mM, while metal chelatingand sulfhydryl reagents did not significantly affect activity. Un-like cytochrome c peroxidases (23) and HRP (6), which arestrongly inhibited by diethyl pyrocarbonate (DEPC) at neutralpH because of the modification of the essential distal histidine,AnaPX was only slightly inhibited (�50% inhibition at 50 mM

DEPC). AnaPX also showed very low sensitivity to KCN, an-other potent peroxidase inhibitor, which caused only 9% and14% inhibition of the activity at 1 and 10 mM, respectively.

Substrate specificity. The activity of AnaPX toward selectedhydrogen donors and aromatic substrates was determined inthe presence of H2O2 and compared with that of HRP undersimilar assay conditions (Table 2). Neither enzyme could oxi-dize Mn(II) and veratryl alcohol, typical manganese peroxidaseand lignin peroxidase substrates, respectively. However,AnaPX exhibited a broader specificity than HRP for L-ascor-bate, 4-aminoantipyrine, pyrogallol, D-iso-ascorbate, NADH,NADPH, and RB5 at pH 4.0. HRP exhibited a distinct pref-erence for guaiacol, 4-aminoantipyrine, and pyrogallol as elec-tron donors under similar conditions. The enzyme also hadmoderate activity toward two guaiacyl lignin unit (G-type)-derived phenolic compounds characterized by two methoxysubstituents in the ortho position to the phenolic hydroxyl,syringaldehyde (54 U mg�1) and 2-DMP (71 U mg�1) (Table2). The activity toward syringaldehyde was associated with adecrease in absorption peak at 320 nm and formation of a newpeak at 315 nm due to product formation (data not shown).Due to overlapping of the substrate and product absorptionpeaks observed in our study, the actual AnaPX activity towardthis substrate may be higher than reported.

Dye decolorization activity of AnaPX. The ability of thepurified AnaPX to decolorize representative synthetic dyes wastested under standard conditions, as described in the Materialsand Methods section. The enzyme efficiently decolorized AQdyes RB5 (262 U mg�1), RB4 (167 U mg�1), RB114 (491 Umg�1), and RB19 (401 U mg�1), with 90% decolorization ofthese dyes obtained within 5 min (Table 3). The enzyme alsodecolorized azo dyes at rates of 8, 13, 91, and 21 U mg�1 forReactive Yellow 86, Reactive Red 120, Reactive Green 19, andReactive Black 5, respectively. Except for Direct Sky Blue 6Band Reactive Green 19, the decolorization activity of AnaPXtoward azo dyes was �20% relative to activity toward RB5.However, AnaPX could not decolorize two azo dyes, namely,

TABLE 2. Substrate specificity of recombinant AnaPX and HRP

Electron donor Concn(mM)

Specific activity (U mg�1)a

AnaPX HRP

Guaiacol 10 230 � 23 (100) 191 � 6 (100)4-Aminoantipyrine 5 1478 � 87 (643) 324 � 51 (169)2,6-Dimethoxyphenol 3 71 � 4 (31) NDABTS 1.5 204 � 12 (89) 968 � 32 (506)Mn(II) 0.4 0 0Veratryl alcohol 0.3 0 0Pyrogallol 20 216 � 27 (93) 238 � 13 (124)RB5 0.1 282 � 12 (123) 28 � 4 (15)L-Ascorbate 0.4 290 � 23 (126) 7 � 0.7 (4)D-Isoascorbate 0.4 308 � 19 (134) 9 � 0.5 (5)NADH 0.15 374 � 5 (163) 13 � 1.2 (7)NADPH 0.04 308 � 13 (134) 3 � 0.7 (2)Syringaldehyde 0.5 54 � 6 (23)

a Values in parentheses are relative activity (%) with the value for guaiacoloxidation set at 100%. ND, not determined.

TABLE 3. Dye decolorization activity of AnaPX against various dyes

Dye Type max Optimal pH Decolorizationrate (U mg�1)a

Decolorization(%)b

RB5 AQ 600 4.8 262 � 13.0 (100) 91 � 5.2RB4 AQ 597 4.4 167 � 5.2 (64) 94 � 2.3RB114 AQ 620 5 491 � 23.1 (187) 96 � 2.8RB19 AQ 590 4.8 401 � 10.5 (153) 94 � 3.2Acid Blue 45 AQ 602 4.6 256 � 26.5 (98) 91 � 5.0Reactive Black 5 Azo 598 4.4 21 � 3.5 (8) 12 � 3.4Reactive Green 19 Azo 622 4 91 � 6.0 (35) 27 � 8.7Reactive Red 120 Azo 535 4 13 � 2.6 (5) 12 � 2.7Reactive Yellow 86 Azo 417 4.2 8 � 1.3 (3) 1 � 0.5Reactive Red 33 Azo 622 4�5 NRD (0) 0Reactive Yellow 2 Azo 390 4�5 NRD (0) 0Reactive Orange 14 Azo 434 4 26 � 4.1 (10) 5 � 0.9Direct Sky Blue 6B Azo 610 4 131 � 16.7 (50) 44 � 8.0Congo Red Azo 512 4.2 32 � 5.0 (12) 4 � 0.5Acid Red 151 Azo 484 4.0 32 � 4.3 (12) 12 � 4.8Procion Blue H-ERD Triazine 618 4.4 236 � 11.4 (90) 80 � 10Procion Blue H-EXL Triazine 628 4.4 188 � 9.0 (72) 72 � 5.6Toluidine Blue O Heterocyclic 620 4�5 NRD (0) 0

a Values in parentheses are relative decolorization rates (%) with the value for RB5 set at 100%. NRD, no reaction detected.b Percent decrease in absorbance of the dye solutions at their respective max values after 2 h.

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Reactive Red 33 and Reactive Yellow 2, and one heterocyclicdye, namely, Toluidine Blue O. We also compared the decol-orization abilities of AnaPX and HRP for various syntheticdyes (Fig. 5). AnaPX more effectively decolorized the dyesstudied than HRP. In particular, AnaPX showed 7.8- and 14-fold activity for RB114 and RB19, respectively. However, bothenzymes were found to be generally ineffective with regard todecolorizing azo dyes.

The AnaPX peroxidase-catalyzed decolorization of azo dyeswas also examined in the presence of syringaldehyde, a lignin-derived mediator compound (Fig. 6). Various concentrations(10 to 800 �M) of syringaldehyde were tested for the mediatoreffect, where a 40 �M concentration gave optimum effect. Thepresence of syringaldehyde drastically enhanced the rate ofdecolorization of Reactive Black 5, Reactive Orange 14, Re-active Red 120, Reactive Green 19, and Acid Red with 50-fold,9-fold, 15-fold, 2-fold, and 7-fold improvements, respectively.Indeed, 0.635 U/ml of AnaPX in the presence of 40 �M syrin-galdehyde at 37°C for 2 h decolorized the abovementioneddyes up to 98%, 69%, 90%, 78%, and 89%, respectively. Incontrast, treatment of HRP with syringaldehyde caused onlyslight improvements in decolorization rates of these dyes withsixfold, twofold, twofold, twofold, and threefold improvements

for Reactive Black 5, Reactive Orange 14, Reactive Red 120,Reactive Green 19, and Acid Red, respectively (data notshown). However, Reactive Brown 10 and Reactive Orange 86were recalcitrant to decolorization, even in the presence ofsyringaldehyde (Fig. 6).

Kinetic parameter analysis. The steady-state kinetics pa-rameters of the AnaPX are summarized in Table 4. The Km

values (H2O2 concentration that gives 50% of the apparentmaximal activity) for H2O2 (5.8 �M) and RB5 (3.6 �M) weresmaller than those reported for other peroxidases. The second-order plots of 1/AnaPX versus l/[H2O2] at various fixed con-centrations of H2O2 yielded a set of parallel lines, indicating aping-pong mechanism for the oxidation of RB5 by AnaPX(data not shown). Additionally, the secondary plot of the pri-mary y-intercepts versus 1/[H2O2] revealed a linear relation-ship, indicating that the AnaPX activity on RB5 involves clas-sical ping-pong bi-bi kinetics.

DISCUSSION

Compared to class I, II, and III peroxidases, DyPs representa large group of hemoproteins that are yet to be understoodalthough they are found in almost all bacterial genomes de-posited in gene databases. The peroxidases encoded by alr1585and its three cyanobacterial homologues do not show any sig-nificant homologies to other bacterial DyPs (Fig. 1b). How-ever, AnaPX and its cyanobacterial homologues share conser-vation of the heme binding site, especially catalyticallyimportant distal and proximal residues. The recent resolutionof the X-ray crystal structures of three DyPs—T. cucumerisDec1 DyP (32), Bacteroides thetaiotoamicron DyP (42), andShewanella oneidensis TyrA (41)—revealed a unique tertiarystructure and distal heme region that differ from those of mostother peroxidases. The catalytically important distal histidineresidue conserved in the typical peroxidases is absent in DyPs;instead, a conserved aspartic acid residue in conjunction witharginine has been proposed to assist in the formation of anFe4� oxoferryl center and a porphyrin cation radical interme-diate (compound I) (32). In AnaPX, Asp204 was predicted tobe the distal aspartate, with Arg365 acting as the essentialactive site arginine.

SDS-PAGE and size exclusion chromatography analysis ofpurified recombinant enzyme revealed that AnaPX is a tet-rameric protein. This feature has been observed in bacterialDyPs that are predominantly oligomeric (29, 41, 42) comparedto the largely monomeric secretory fungal DyPs (13, 15, 18).

FIG. 5. Decolorization of selected dyes by AnaPX (filled bars) andHRP (open bars) under standard reaction conditions.

FIG. 6. Enhancement of AnaPX decolorization activities on azodyes in the presence of a natural mediator compound, syringaldehyde(40 �M). The decolorization rate in the absence of mediator was set at100%.

TABLE 4. Steady-state kinetic parameters of AnaPX and otherperoxidases for RB5 and H2O2

a

PeroxidaseKm (�M)

kcat(s�1)kcat/Km (M�1 s�1)

RB5 H2O2 RB5 H2O2

AnaPX 3.6 5.8 384 1.2 � 107 6.6 � 106

HRP 58 36 140 2.4 � 106 3.8 � 106

DyP 54 26 260 4.8 � 106 1.0 � 107

VP 4.0 5–10 4.0 1.0 � 106 NDTyrA 84 ND 5.9 7.0 � 105 ND

a Kinetic constants were obtained from the literature in the case of HRP andT. cucumeris Dec1 DyP (18), P. eryngii versatile peroxidase (VP) (4), and S.onediensis TyrA DyP (41). ND, not determined.

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AnaPX, like most members of the DyP family, exhibited anacidic isoelectric point (pI) of 3.68, which does not differ fromthat reported for T. cucumeris Dec1 DyP (3.8) (18) or theMsP1 (3.1) and MsP2 (3.7) isozymes of M. scorodonius perox-idase (27). However, AnaPX exhibited slightly higher pH op-tima (pH 4.0 to 4.4) (Fig. 3b) than T. cucumeris Dec1 DyP (pH3.0 to 3.2).

The absorption maxima of AnaPX were comparable to thoseof high-spin heme peroxidases (24, 39). In the presence ofcyanide, conversion of the ferric high-spin state to the low-spinstate was observed in AnaPX. The absorption patterns were inagreement with those of low-spin, hexacoordinate species ofperoxidases with histidine as the fifth ligand (24, 39), suggest-ing that the fifth ligand to the heme iron of AnaPX is ahistidine residue. The oxidized species of AnaPX formed in thepresence of H2O2 also showed absorption spectra typical of acompound I product.

Although less stable at 50°C and 60°C, AnaPX showed highstability at 30°C and 40°C (Fig. 3a). The enzyme maintained83% and 78% residual activities when kept at 30°C and 40°C,respectively, for 4 days. The stabilities of AnaPX and HRPwere also compared by exposing them to different H2O2 con-centrations (Fig. 3d). Both peroxidases exhibited similar decaycurves as the H2O2 concentration increased, with a 90% lossin activity at 15 mM. This indicates low stability at high per-oxide concentrations. This is consistent with other peroxidases;it appears that an excess of H2O2 causes the irreversible con-version from compound II to inactive compound III, which inturn decreases activity. However, AnaPX exhibited a relativelyhigher tolerance than HRP at similar H2O2 concentrations(Fig. 3d). AnaPX also showed different inhibition profiles inthe presence of various inhibitor compounds. Unlike cyto-chrome c peroxidases (23) and HRP (6), which are stronglyinhibited by DEPC at neutral pH because of the modificationof the essential distal histidine, AnaPX was only slightly inhib-ited (�50% inhibition at 50 mM DEPC). Therefore, the dif-ferences in the inhibition profiles of AnaPX and other plant-type peroxidases in the presence of KCN, NaN3, and DEPCmay be attributable to the differences in the accessibility of theheme active-site and catalytic residues.

Peroxidases are generally specific for H2O2 as the substratebut can use a number of hydrogen donors. AnaPX can oxidizeboth phenolic compounds such as guaiacol, 4-aminoantipyrine,and 2,6-dimethoxyphenol with activities of 230, 1,478, and 71 Umg�1, respectively, more effectively than HRP. Remarkably,the enzyme could also oxidize L-ascorbate and D-isoascorbate,which are typical ascorbate peroxidase substrates. Comparedto the T. cucumeris Dec1 DyP1 isozyme, which shows 1%oxidative activities of both guaiacol and 2,6-DMP relative toRB5 activity (33), AnaPX showed high activity for the twosubstrates. Since the natural substrate of AnaPX remains un-known, the specific peroxidase activity may be significantlyhigher in vivo. AnaPX could also effectively degrade variousAQ dyes compared to HRP (Fig. 5). In particular, AnaPXshowed more effective decolorization activities (90%) towardRB19 (Remazol brilliant blue R) and RB114 (Drimarene bril-liant blue K-BL) with a vinyl sulfonic reactive moiety in theirstructures (Table 3); these dyes are generally resistant to chem-ical oxidation because their aromatic anthracene-9,10-dionestructure is highly stabilized by resonance. In addition, the

enzyme showed higher decolorization activity toward RB5,RB4, Acid Blue 45, and the triazine dyes Procion Blue H-ERDand Procion Blue H-EXL, with 70% decolorization within2 h. Decolorization of RB5 and Acid Blue 45 by AnaPX re-sulted in a decrease in absorbance at 600 nm and an increasein absorbances at 400 to 500 nm accompanied with formationof a reddish-brown product with an azo link (2,2-disulfonylazobenzene). These findings are consistent with those reportedfor the DyP1 isozyme (31). Interestingly, the formation ofreddish-brown product was not observed in the vinyl sulfonateAQ dyes (RB19, RB114, and RB4), indicating that AnaPXuses different degradative pathways for these dyes and for RB5and Acid Blue 45. The apparent AnaPX affinity to H2O2

(Km 5.8 �M) and RB5 (Km 3.6 �M) was higher than thatreported for the partially purified native T. cucumeris Dec1DyP and HRP (18) but was in the same range as the affinity ofthe Pleurotus eryngii versatile peroxidase (4). The kinetic pa-rameters determined for AnaPX clearly revealed that it has ahigher affinity and greater redox potential than HRP and otherperoxidases for H2O2 and RB5. Indeed, this may explainAnaPX’s higher decolorization activity toward RB5. Thus, thisenzyme appears to be unique because it has broader substratespecificity in addition to dye decolorization activity than T.cucumeris Dec1 DyP, which shows higher activity only towardAQ dyes rather than toward a typical substrate like guaiacol.

In contrast to AQ dyes, AnaPX weakly decolorized azo dyes,a property also observed in other DyP enzymes (13, 18). Themajority of azo dyes, due to presence of azo linkages (R-N N-R�) in their structure, are generally recalcitrant to the action ofoxidoreductive enzymes including peroxidases and laccases;however, the presence of redox mediators can significantlyimprove their decolorization (8, 11, 22). In our study, thedecolorization range and oxidation rates of AnaPX comparedto HRP for azo dyes were also drastically enhanced in thepresence of a phenolic redox mediator, syringaldehyde. Therole of redox mediators in the laccase oxidation reaction is nowwell characterized, and redox mechanisms have been pro-posed. The cationic radical is involved in either one-electronoxidation of the substrate to a radical cation or abstraction ofa proton from the substrate, converting it into a radical (7, 37).It is very likely the phenoxy radicals formed during oxidation ofsyringaldehyde by AnaPX have similar properties to those ofthe laccase-mediator system; however, the actual fate and pos-sible reactions of the mediator itself need further clarification.Compared to synthetic mediators that have the drawbacks ofcost and potential toxicity, however, syringaldehyde is a poten-tially promising natural and cheap mediator for industrial ap-plication of AnaPX and other DyP-type enzymes, particularlyin the bioremediation of azo dyes.

The AnaPX structural model showed a conserved tertiarystructure of a two-domain, ��� protein, with each domainconsisting of a four-stranded, antiparallel �-sheet sandwichedby �-helices in a ferredoxin-like fold (32, 42). In addition, theheme is sandwiched between the proximal and distal domains,with the heme edge relatively inaccessible. However, AnaPXand other DyPs possess a “funnel” or V-shaped channel ex-tending �20 Å from the surface opening directly on the distalside of the heme pocket between catalytic residues Asp204 andArg365 (Fig. 7). This channel presumably permits the trans-port of smaller aromatic substrates and peroxides into and out

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of the active site but may also form the substrate-binding sitefor large substrate molecules in DyPs. The size and hydropho-bicity of this substrate channel in AnaPX model were relativelysimilar to the size and hydrophobicity of the T. cucumeris Dec1DyP structure, with the AnaPX channel having 468 and 65total contact and hydrophobic contact atoms, respectively,while the T. cucumeris Dec1 DyP channel consists of 424 and62 total contact and hydrophobic contact atoms, respectively.We performed docking simulation of guaiacol (a small aro-matic substrate) and RB5 (a bulkier substrate) using the ASEDockfunction of the MOE software to better understand the sub-strate binding and access modes in these two proteins. Asshown in Fig. 7b, both proteins showed similar binding levels ofRB5, with the anthraquinone moiety oriented �5 Å toward theheme pocket entrance; that may explain the similar reactivitiesof AnaPX and the fungal DyP toward RB5. However, theseproteins showed different accessibilities of guaiacol to the ac-tive site (Fig. 7b). In the AnaPX model, guaiacol easily ac-cesses the active site and binds �2.5 Å from the iron centerwithin the heme pocket. In contrast, the fungal DyP has aconstricted heme pocket entrance that provides steric hin-drance, limiting accessibility of substrates, and thus the guaia-col molecule binds within a hydrogen bond distance to catalyticresidues Asp171 and Arg329 outside the heme pocket. AnaPXshows relatively higher activities toward both guaiacol andRB5 than T. cucumeris Dec1 DyP, which shows a higher spec-ificity toward AQ than guaiacol that may be attributable todifferent accessibilities of the guaiacol molecule to the hemeactive site, as deduced by the above docking experiments. Thesubstrate access channel of both proteins was also inaccessibleto Reactive Black 5, which is bulkier than RB5, which mayexplain the low reactivity of DyPs toward azo dyes. It is notablethat DyPs utilize different substrate binding sites from typicalperoxidases, and their broad activities characterized thus farmay suggest the involvement of unique radicals or an activeoxygen species. In addition, due to the multiple oligomeriza-tion states exhibited by AnaPX, it was unclear whether oli-gomerization also affects the substrate channel and substrate

specificity in contrast to the monomeric fungal DyPs. However,these hypotheses based on in silico analysis require furtherstudy.

In conclusion, we have shown that the alr1585 in theAnabaena sp. strain PCC 7120 genome encodes a novel heme-dependent peroxidase. The enzyme shares certain unique mo-lecular characteristic with fungal DyP proteins. Its low pHoptima, enzymatic properties, and broad specificity for aro-matic compounds and recalcitrant synthetic dyes make AnaPXa versatile DyP. The enlarged substrate spectrum of AnaPXmay open new possibilities for biotechnological applications ofDyP-type peroxidases, including bioremediation of wastewatercontaminated by xenobiotic compounds. Furthermore, its highlevel of activity toward peroxidase substrates in addition to itshigh overproduction in recombinant hosts indicates thatAnaPX could be a useful alternative to HRP or fungal DyP inbiotechnological or bioindustrial applications. It may be thatthe same peculiarity in structure, including the nature of theaxial ligands and the environment of the substrate-binding sitein AnaPX, is also responsible for the other observed differ-ences between the DyPs and the plant superfamily peroxidases.Further physicochemical and kinetic studies of DyPs are pres-ently being carried out in our laboratory to better understandhow the protein environment modulates the activity of theheme.

ACKNOWLEDGMENTS

We are grateful to Katsuyuki Tanizawa of the Institute of Scientificand Industrial Research, Osaka University, for the kind gift of an E.coli BL21(DE3) strain with disrupted catalase genes (katE and katG).

REFERENCES

1. Abadulla, E., T. Tzanov, S. Costa, K. H. Robra, A. Cavaco-Paulo, and G. M.Gubitz. 2000. Decolorization and detoxification of textile dyes with a laccasefrom Trametes hirsuta. Appl. Environ. Microbiol. 66:3357–3362.

2. Ausubel, F. M. 2002. Short protocols in molecular biology: a compendium ofmethods from Current Protocols in Molecular Biology, 5th ed. Wiley, NewYork, NY.

3. Bafana, A., T. Chakrabarti, and S. S. Devi. 2008. Azoreductase and dyedetoxification activities of Bacillus velezensis strain AB. Appl. Microbiol.Biotechnol. 77:1139–1144.

FIG. 7. Homology model of AnaPX showing the substrate access channel. (a) Structural alignment of AnaPX model (light blue) and T.cucumeris Dec1 DyP backbone (blue) showing the Gaussian contact surface model of the V-shaped substrate access channel opening to the hemepocket and heme (red). (b) Binding of RB5 and guaiacol in the substrate access channel in the AnaPX model (G1) and T. cucumeris Dec1 DyP(G2). The AnaPX heme and active site are depicted as sticks while the corresponding T. cucumeris Dec1 DyP residues (in parentheses) arepresented as red lines.

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4. Banci, L., S. Camarero, A. T. Martinez, M. J. Martinez, M. Perez-Boada, R.Pierattelli, and F. J. Ruiz-Duenas. 2003. NMR study of manganese(II)binding by a new versatile peroxidase from the white-rot fungus Pleurotuseryngii. J. Biol. Inorg. Chem. 8:751–760.

5. Berry, E. A., and B. L. Trumpower. 1987. Simultaneous determination ofhemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem. 161:1–15.

6. Bhattacharyya, D. K., U. Bandyopadhyay, and R. K. Banerjee. 1993. Chem-ical and kinetic evidence for an essential histidine residue in the electrontransfer from aromatic donor to horseradish peroxidase compound I. J. Biol.Chem. 268:22292–22298.

7. Caldwell, E. S., and C. Steelink. 1969. Phenoxy radical intermediates in theenzymatic degradation of lignin model compounds. Biochim. Biophys. Acta184:420–431.

8. Camarero, S., D. Ibarra, M. J. Martinez, and A. T. Martinez. 2005. Lignin-derived compounds as efficient laccase mediators for decolorization of dif-ferent types of recalcitrant dyes. Appl. Environ. Microbiol. 71:1775–1784.

9. Chivukula, M., and V. Renganathan. 1995. Phenolic azo dye oxidation bylaccase from Pyricularia oryzae. Appl. Environ. Microbiol. 61:4374–4377.

10. Christian, V. V., R. Shrivastava, C. Novotny, and B. R. Vyas. 2003. Decol-orization of sulfonphthalein dyes by manganese peroxidase activity of thewhite-rot fungus Phanerochaete chrysosporium. Folia Microbiol. (Praha) 48:771–774.

11. Claus, H., G. Faber, and H. Konig. 2002. Redox-mediated decolorization ofsynthetic dyes by fungal laccases. Appl. Microbiol. Biotechnol. 59:672–678.

12. Dey, S., T. K. Maiti, and B. C. Bhattacharyya. 1994. Production of someextracellular enzymes by a lignin peroxidase-producing brown rot fungus,Polyporus ostreiformis, and its comparative abilities for lignin degradationand dye decolorization. Appl. Environ. Microbiol. 60:4216–4218.

13. Faraco, V., A. Piscitelli, G. Sannia, and P. Giardina. 2006. Identification ofa new member of the dye-decolorizing peroxidase family from Pleurotusostreatus. World J. Microbiol. Biotechnol. 23:889–893.

14. Golden, J. W., and H. S. Yoon. 2003. Heterocyst development in Anabaena.Curr. Opin. Microbiol. 6:557–563.

15. Johjima, T., M. Ohkuma, and T. Kudo. 2003. Isolation and cDNA cloning ofnovel hydrogen peroxide-dependent phenol oxidase from the basidiomyceteTermitomyces albuminosus. Appl. Microbiol. Biotechnol. 61:220–225.

16. Kamitsuji, H., Y. Honda, T. Watanabe, and M. Kuwahara. 2004. Productionand induction of manganese peroxidase isozymes in a white-rot fungus Pleu-rotus ostreatus. Appl. Microbiol. Biotechnol. 65:287–294.

17. Kaneko, T., Y. Nakamura, C. P. Wolk, T. Kuritz, S. Sasamoto, A. Watanabe,M. Iriguchi, A. Ishikawa, K. Kawashima, T. Kimura, Y. Kishida, M. Kohara,M. Matsumoto, A. Matsuno, A. Muraki, N. Nakazaki, S. Shimpo, M. Sugi-moto, M. Takazawa, M. Yamada, M. Yasuda, and S. Tabata. 2001. Completegenomic sequence of the filamentous nitrogen-fixing cyanobacteriumAnabaena sp. strain PCC 7120. DNA Res. 8:205–213, 227–253.

18. Kim, S. J., and M. Shoda. 1999. Purification and characterization of a novelperoxidase from Geotrichum candidum Dec 1 involved in decolorization ofdyes. Appl. Environ. Microbiol. 65:1029–1035.

19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

20. Levitt, M. 1992. Accurate modeling of protein conformation by automaticsegment matching. J. Mol. Biol. 226:507–533.

21. Luthy, R., J. U. Bowie, and D. Eisenberg. 1992. Assessment of proteinmodels with three-dimensional profiles. Nature 356:83–85.

22. Matto, M., and Q. Husain. 2007. Decolorization of direct dyes by salt frac-tionated turnip proteins enhanced in the presence of hydrogen peroxide andredox mediators. Chemosphere 69:338–345.

23. McGinnity, D. F., B. Devreese, S. Prazeres, J. Van Beeumen, I. Moura, J. J.Moura, and G. W. Pettigrew. 1996. A single histidine is required for activityof cytochrome c peroxidase from Paracoccus denitrificans. J. Biol. Chem.271:11126–11133.

24. Mino, Y., H. Wariishi, N. J. Blackburn, T. M. Loehr, and M. H. Gold. 1988.Spectral characterization of manganese peroxidase, an extracellular hemeenzyme from the lignin-degrading basidiomycete, Phanerochaete chrysospo-rium. J. Biol. Chem. 263:7029–7036.

25. Ollikka, P., K. Alhonmaki, V. M. Leppanen, T. Glumoff, T. Raijola, and I.Suominen. 1993. Decolorization of azo, triphenyl methane, heterocyclic, andpolymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysos-porium. Appl. Environ. Microbiol. 59:4010–4016.

26. Robinson, T., G. McMullan, R. Marchant, and P. Nigam. 2001. Remediationof dyes in textile effluent: a critical review on current treatment technologieswith a proposed alternative. Bioresour. Technol. 77:247–255.

27. Scheibner, M., B. Hulsdau, K. Zelena, M. Nimtz, L. de Boer, R. G. Berger,and H. Zorn. 2008. Novel peroxidases of Marasmius scorodonius degradebeta-carotene. Appl. Microbiol. Biotechnol. 77:1241–1250.

28. Shimokawa, T., M. Shoda, and Y. Sugano. 2009. Purification and character-ization of two DyP isozymes from Thanatephorus cucumeris Dec 1 specificallyexpressed in an air-membrane surface bioreactor. J. Biosci. Bioeng. 107:113–115.

29. Sturm, A., A. Schierhorn, U. Lindenstrauss, H. Lilie, and T. Bruser. 2006.YcdB from Escherichia coli reveals a novel class of Tat-dependently trans-located hemoproteins. J. Biol. Chem. 281:13972–13978.

30. Sugano, Y. 2009. DyP-type peroxidases comprise a novel heme peroxidasefamily. Cell Mol. Life Sci. 66:1387–1403.

31. Sugano, Y., Y. Matsushima, K. Tsuchiya, H. Aoki, M. Hirai, and M. Shoda.2009. Degradation pathway of an anthraquinone dye catalyzed by a uniqueperoxidase DyP from Thanatephorus cucumeris Dec 1. Biodegradation 20:433–440.

32. Sugano, Y., R. Muramatsu, A. Ichiyanagi, T. Sato, and M. Shoda. 2007. DyP,a unique dye-decolorizing peroxidase, represents a novel heme peroxidasefamily: ASP171 replaces the distal histidine of classical peroxidases. J. Biol.Chem. 282:36652–36658.

33. Sugano, Y., R. Nakano, K. Sasaki, and M. Shoda. 2000. Efficient heterolo-gous expression in Aspergillus oryzae of a unique dye-decolorizing peroxidase,DyP, of Geotrichum candidum Dec 1. Appl. Environ. Microbiol. 66:1754–1758.

34. Sugano, Y., K. Sasaki, and M. Shoda. 1999. cDNA cloning and geneticanalysis of a novel decolorizing enzyme, peroxidase gene dyp from Geotri-chum candidum Dec 1. J. Biosci. Bioeng. 87:411–417.

35. Vandevivere, P. C., R. Bianchi, and W. Verstraete. 1998. Treatment andreuse of wastewater from the textile wet-processing industry: review ofemerging technologies. J. Chem. Technol. Biotechnol. 72:289–302.

36. Verma, P., and D. Madamwar. 2002. Production of ligninolytic enzymes fordye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus andPhanerochaete chrysosporium under solid-state fermentation. Appl. Biochem.Biotechnol. 102–103:109–118.

37. Xu, F., H. J. Deussen, B. Lopez, L. Lam, and K. Li. 2001. Enzymatic andelectrochemical oxidation of N-hydroxy compounds. Redox potential, elec-tron-transfer kinetics, and radical stability. Eur. J. Biochem. 268:4169–4176.

38. Yang, Q., M. Yang, K. Pritsch, A. Yediler, A. Hagn, M. Schloter, and A.Kettrup. 2003. Decolorization of synthetic dyes and production of manga-nese-dependent peroxidase by new fungal isolates. Biotechnol. Lett. 25:709–713.

39. Youn, H. D., Y. I. Yim, K. Kim, Y. C. Hah, and S. O. Kang. 1995. Spectralcharacterization and chemical modification of catalase-peroxidase fromStreptomyces sp. J. Biol. Chem. 270:13740–13747.

40. Zille, A., B. Gornacka, A. Rehorek, and A. Cavaco-Paulo. 2005. Degradationof azo dyes by Trametes villosa laccase over long periods of oxidative condi-tions. Appl. Environ. Microbiol. 71:6711–6718.

41. Zubieta, C., R. Joseph, S. S. Krishna, D. McMullan, M. Kapoor, H. L.Axelrod, M. D. Miller, P. Abdubek, C. Acosta, T. Astakhova, D. Carlton,H. J. Chiu, T. Clayton, M. C. Deller, L. Duan, Y. Elias, M. A. Elsliger, J.Feuerhelm, S. K. Grzechnik, J. Hale, G. W. Han, L. Jaroszewski, K. K. Jin,H. E. Klock, M. W. Knuth, P. Kozbial, A. Kumar, D. Marciano, A. T. Morse,K. D. Murphy, E. Nigoghossian, L. Okach, S. Oommachen, R. Reyes, C. L.Rife, P. Schimmel, C. V. Trout, H. van den Bedem, D. Weekes, A. White, Q.Xu, K. O. Hodgson, J. Wooley, A. M. Deacon, A. Godzik, S. A. Lesley, andI. A. Wilson. 2007. Identification and structural characterization of hemebinding in a novel dye-decolorizing peroxidase, TyrA. Proteins 69:234–243.

42. Zubieta, C., S. S. Krishna, M. Kapoor, P. Kozbial, D. McMullan, H. L.Axelrod, M. D. Miller, P. Abdubek, E. Ambing, T. Astakhova, D. Carlton,H. J. Chiu, T. Clayton, M. C. Deller, L. Duan, M. A. Elsliger, J. Feuerhelm,S. K. Grzechnik, J. Hale, E. Hampton, G. W. Han, L. Jaroszewski, K. K. Jin,H. E. Klock, M. W. Knuth, A. Kumar, D. Marciano, A. T. Morse, E. Nigog-hossian, L. Okach, S. Oommachen, R. Reyes, C. L. Rife, P. Schimmel, H. vanden Bedem, D. Weekes, A. White, Q. Xu, K. O. Hodgson, J. Wooley, A. M.Deacon, A. Godzik, S. A. Lesley, and I. A. Wilson. 2007. Crystal structures oftwo novel dye-decolorizing peroxidases reveal a beta-barrel fold with a con-served heme-binding motif. Proteins 69:223–233.

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