Structural and Functional Features of Peroxidases with a Potential as Industrial Biocatalysts

  • Francisco J. Ruiz-Dueñas
  • Angel T. Martínez


This chapter begins with a description of the main structural features of heme peroxidases representative of the two large superfamilies of plant–fungal–bacterial and animal peroxidases, and the four additional (super)families described to date. Then, we focus on several fungal peroxidases of high biotechnological potential as industrial biocatalysts. These include (1) ligninolytic peroxidases from white-rot basidiomycetes being able to oxidize high redox-potential substrates at an exposed protein radical; (2) heme-thiolate peroxidases that are structural hybrids of typical peroxidases and cytochrome P450 enzymes and, after their discovery in sooty molds, are being described in basidiomycetes with even more interesting catalytic properties, such as selective aromatic oxygenation; and (3) the so-called dye-decolorizing peroxidases that are still to be thoroughly investigated but have been identified in different basidiomycete genomes. The structural–functional description of these peroxidases includes an analysis of the heme environment and a description of their substrate oxidation sites, with the purpose of understanding their interesting catalytic properties and biotechnological potential.


Versatile Peroxidase Heme Pocket Biotechnological Interest Heme Peroxidase Distal Histidine 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors thank Martin Hofrichter (University of Zittau, Germany) for useful information on basidiomycete heme-thiolate and DyP-type peroxidases. The financial support of the BIORENEW EU-contract on “White Biotechnology for added value products from renewable plant polymers: Design of tailor-made biocatalyst and new industrial bioprocesses” (NMP2-CT-2006-026456) and the RAPERO Spanish Biotechnology project on “Radical peroxidases” (BIO2008-01533) is acknowledged. F.J.R.-D. thanks the Spanish MICINN for a “Ramón y Cajal” contract.


  1. 1.
    Finzel BC, Poulos TL, Kraut J (1984) Crystal structure of yeast cytochrome c peroxidase refined at 1.7 Å resolution. J Biol Chem 259:13027–13036Google Scholar
  2. 2.
    Koua D, Cerutti L, Falquet L et al (2009) PeroxiBase: a database with new tools for peroxidase family classification. Nucleic Acids Res 37:D261–D266CrossRefGoogle Scholar
  3. 3.
    Welinder KG (1992) Superfamily of plant, fungal and bacterial peroxidases. Curr Opin Struct Biol 2:388–393CrossRefGoogle Scholar
  4. 4.
    Welinder KG, Gajhede M (1993) Structure and evolution of peroxidases. In: Greppin H, Rasmussen SK, Welinder KG et al (eds) Plant peroxidases: biochemistry and physiology. University of Copenhagen and University of Geneve, Geneve, pp 35–42Google Scholar
  5. 5.
    Dunford HB (1999) Heme peroxidases. Wiley, New YorkGoogle Scholar
  6. 6.
    Banci L (1997) Structural properties of peroxidases. J Biotechnol 53:253–263CrossRefGoogle Scholar
  7. 7.
    Sharp KH, Mewies M, Moody PC et al (2003) Crystal structure of the ascorbate peroxidase-ascorbate complex. Nat Struct Biol 10:303–307CrossRefGoogle Scholar
  8. 8.
    Sundaramoorthy M, Youngs HL, Gold MH et al (2005) High-resolution crystal structure of manganese peroxidase: substrate and inhibitor complexes. Biochemistry 44:6463–6470CrossRefGoogle Scholar
  9. 9.
    Ruiz-Dueñas FJ, Morales M, Pérez-Boada M et al (2007) Manganese oxidation site in Pleurotus eryngii versatile peroxidase: A site-directed mutagenesis, kinetic and crystallographic study. Biochemistry 46:66–77CrossRefGoogle Scholar
  10. 10.
    Ruiz-Dueñas FJ, Morales M, García E et al (2009) Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases. J Exp Bot 60:441–452CrossRefGoogle Scholar
  11. 11.
    Kimura S, Ikeda-Saito M (1988) Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family. Proteins 3:113–120CrossRefGoogle Scholar
  12. 12.
    Zeng J, Fenna RE (1992) X-ray crystal-structure of canine myeloperoxidase at 3 Å resolution. J Mol Biol 226:185–207CrossRefGoogle Scholar
  13. 13.
    Fiedler TJ, Davey CA, Fenna RE (2000) X-ray crystal structure and characterization of halide-binding sites of human myeloperoxidase at 1.8 angstrom resolution. J Biol Chem 275:11964–11971CrossRefGoogle Scholar
  14. 14.
    Singh AK, Singh N, Sharma S et al (2008) Crystal structure of lactoperoxidase at 2.4 angstrom resolution. J Mol Biol 376:1060–1075CrossRefGoogle Scholar
  15. 15.
    Zederbauer M, Furtmuller PG, Brogioni S et al (2007) Heme to protein linkages in mammalian peroxidases: impact on spectroscopic, redox and catalytic properties. Nat Prod Rep 24:571–584CrossRefGoogle Scholar
  16. 16.
    Huang LS, Ortiz de Montellano PR (2006) Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation. Arch Biochem Biophys 446:77–83CrossRefGoogle Scholar
  17. 17.
    Huang LS, Wojciechowski G, Ortiz de Montellano PR (2006) Role of heme-protein covalent bonds in mammalian peroxidases – Protection of the heme by a single engineered heme-protein link in horseradish peroxidase. J Biol Chem 281:18983–18988CrossRefGoogle Scholar
  18. 18.
    Furtmüller PG, Zederbauer M, Jantschko W et al (2006) Active site structure and catalytic mechanisms of human peroxidases. Arch Biochem Biophys 445:199–213CrossRefGoogle Scholar
  19. 19.
    Chelikani P, Fita I, Loewen PC (2004) Diversity of structures and properties among catalases. Cell Mol Life Sci 61:192–208CrossRefGoogle Scholar
  20. 20.
    Vainshtein BK, Melikadamyan WR, Barynin VV et al (1986) 3-Dimensional structure of catalase from Penicillium vitale at 2.0 Å resolution. J Mol Biol 188:49–61CrossRefGoogle Scholar
  21. 21.
    Vainshtein BK, Melikadamyan WR, Barynin VV et al (1981) 3-Dimensional structure of the enzyme catalase. Nature 293:411–412CrossRefGoogle Scholar
  22. 22.
    Mate MJ, Zamocky M, Nykyri LM et al (1999) Structure of catalase-A from Saccharomyces cerevisiae. J Mol Biol 286:135–149CrossRefGoogle Scholar
  23. 23.
    Kirkman HN, Galiano S, Gaetani GF (1987) The function of catalase-bound NADPH. J Biol Chem 262:660–666Google Scholar
  24. 24.
    Fülöp V, Ridout CJ, Greenwood C et al (1995) Crystal-structure of the di-heme cytochrome-c peroxidase from Pseudomonas aeruginosa. Structure 3:1225–1233CrossRefGoogle Scholar
  25. 25.
    Dias JM, Alves T, Bonifacio C et al (2004) Structural basis for the mechanism of Ca2+ activation of the di-heme cytochrome c peroxidase from Pseudomonas nautica 617. Structure 12:961–973CrossRefGoogle Scholar
  26. 26.
    De Smet L, Savvides SN, Van Horen E et al (2006) Structural and mutagenesis studies on the cytochrome c peroxidase from Rhodobacter capsulatus provide new insights into structure-function relationships of bacterial di-heme peroxidases. J Biol Chem 281:4371–4379CrossRefGoogle Scholar
  27. 27.
    Sundaramoorthy M, Terner J, Poulos TL (1995) The crystal structure of chloroperoxidase: A heme peroxidase-cytochrome P450 functional hybrid. Structure 3:1367–1377CrossRefGoogle Scholar
  28. 28.
    Sugano Y, Muramatsu R, Ichiyanagi A et al (2007) DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family. J Biol Chem 282:36652–36658CrossRefGoogle Scholar
  29. 29.
    Sugano Y (2009) DyP-type peroxidases comprise a novel heme peroxidase family. Cell Mol Life Sci 66:1387–1403CrossRefGoogle Scholar
  30. 30.
    Ruiz-Dueñas FJ, Martínez AT (2009) Microbial degradation of lignin: How a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this. Microb Biotechnol 2:164–177CrossRefGoogle Scholar
  31. 31.
    Kirk TK, Farrell RL (1987) Enzymatic “combustion”: The microbial degradation of lignin. Annu Rev Microbiol 41:465–505CrossRefGoogle Scholar
  32. 32.
    Kersten P, Cullen D (2007) Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium. Fungal Genet Biol 44:77–87CrossRefGoogle Scholar
  33. 33.
    Martínez AT (2002) Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzyme Microb Technol 30:425–444CrossRefGoogle Scholar
  34. 34.
    Martínez AT (2007) High redox potential peroxidases. In: Polaina J, MacCabe AP (eds) Industrial enzymes: structure, function and applications. Springer, Berlin, pp 475–486Google Scholar
  35. 35.
    Hammel KE, Cullen D (2008) Role of fungal peroxidases in biological ligninolysis. Curr Opin Plant Biol 11:349–355CrossRefGoogle Scholar
  36. 36.
    Martínez D, Challacombe J, Morgenstern I et al (2009) Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc Natl Acad Sci USA 106:1954–1959CrossRefGoogle Scholar
  37. 37.
    Martínez AT, Ruiz-Dueñas FJ, Martínez MJ et al (2009) Enzymatic delignification of plant cell wall: from nature to mill. Curr Opin Biotechnol 20:348–357CrossRefGoogle Scholar
  38. 38.
    Bumpus JA, Tien M, Wright D et al (1985) Oxidation of persistent environmental pollutants by a white rot fungus. Science 228:1434–1436CrossRefGoogle Scholar
  39. 39.
    Rodríguez E, Nuero O, Guillén F et al (2004) Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase. Soil Biol Biochem 36:909–916CrossRefGoogle Scholar
  40. 40.
    Dávila-Vázquez G, Tinoco R, Pickard MA et al (2005) Transformation of halogenated pesticides by versatile peroxidase from Bjerkandera adusta. Enzyme Microb Technol 36:223–231CrossRefGoogle Scholar
  41. 41.
    Wang YX, Vázquez-Duhalt R, Pickard MA (2003) Manganese-lignin peroxidase hybrid from Bjerkandera adusta oxidizes polycyclic aromatic hydrocarbons more actively in the absence of manganese. Can J Microbiol 49:675–682CrossRefGoogle Scholar
  42. 42.
    Heinfling A, Martínez MJ, Martínez AT et al (1998) Transformation of industrial dyes by manganese peroxidase from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction. Appl Environ Microbiol 64:2788–2793Google Scholar
  43. 43.
    Tamura K, Dudley J, Nei M et al (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefGoogle Scholar
  44. 44.
    Larrondo L, Gonzalez A, Perez AT et al (2005) The nop gene from Phanerochaete chrysosporium encodes a peroxidase with novel structural features. Biophys Chem 116:167–173CrossRefGoogle Scholar
  45. 45.
    Ullrich R, Hofrichter M (2007) Enzymatic hydroxylation of aromatic compounds. Cell Mol Life Sci 64:271–293CrossRefGoogle Scholar
  46. 46.
    Ullrich R, Hofrichter M (in press) Novel trends in fungal biooxidation. The Mycota X: Industrial applications. Springer, BerlinGoogle Scholar
  47. 47.
    Kim SJ, Shoda M (1999) Purification and characterization of a novel peroxidase from Geotrichum candidum Dec 1 involved in decolorization of dyes. Appl Environ Microbiol 65:1029–1035Google Scholar
  48. 48.
    Miyazaki-Imamura C, Oohira K, Kitagawa R et al (2003) Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase. Protein Eng 16:423–428CrossRefGoogle Scholar
  49. 49.
    Reading NS, Aust SD (2001) Role of disulfide bonds in the stability of recombinant manganese peroxidase. Biochemistry 40:8161–8168CrossRefGoogle Scholar
  50. 50.
    Miyazaki C, Takahashi H (2001) Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2. FEBS Lett 509:111–114CrossRefGoogle Scholar
  51. 51.
    Reading NS, Aust SD (2000) Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability. Biotechnol Progr 16:326–333CrossRefGoogle Scholar
  52. 52.
    Ruiz-Dueñas FJ, Morales M, Rencoret J et al (2008) Improved peroxidases (Peroxidasas mejoradas). Patent (Spain) P200801292Google Scholar
  53. 53.
    García E, Martínez MJ, Ruiz-Dueñas FJ et al (2009) High redox-potential peroxidases from directed evolution (Peroxidasas de elevado potencial redox diseñadas por evolución dirigida). Patent (Spain) application P200930157Google Scholar
  54. 54.
    Hiner ANP, Raven EL, Thorneley RNF et al (2002) Mechanisms of compound I formation in heme peroxidases. J Inorg Biochem 91:27–34CrossRefGoogle Scholar
  55. 55.
    Banci L, Bertini I, Turano P et al (1991) Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase. Proc Natl Acad Sci USA 88:6956–6960CrossRefGoogle Scholar
  56. 56.
    Gold MH, Youngs HL, Gelpke MD (2000) Manganese peroxidase. Met Ions Biol Syst 37:559–586Google Scholar
  57. 57.
    Pérez-Boada M, Ruiz-Dueñas FJ, Pogni R et al (2005) Versatile peroxidase oxidation of high redox potential aromatic compounds: Site-directed mutagenesis, spectroscopic and crystallographic investigations of three long-range electron transfer pathways. J Mol Biol 354:385–402CrossRefGoogle Scholar
  58. 58.
    Pogni R, Baratto MC, Teutloff C et al (2006) A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multi-frequency EPR and DFT study. J Biol Chem 281:9517–9526CrossRefGoogle Scholar
  59. 59.
    Ruiz-Dueñas FJ, Pogni R, Morales M et al (2009) Protein radicals in fungal versatile peroxidase: catalytic tryptophan radical in both Compound I and Compound II and studies on W164Y, W164H and W164S variants. J Biol Chem 284:7986–7994CrossRefGoogle Scholar
  60. 60.
    Sundaramoorthy M, Kishi K, Gold MH et al (1997) Crystal structures of substrate binding site mutants of manganese peroxidase. J Biol Chem 272:17574–17580CrossRefGoogle Scholar
  61. 61.
    Youngs HL, Gelpke MDS, Li DM et al (2001) The role of Glu39 in Mn-II binding and oxidation by manganese peroxidase from Phanerochaete chrysosporium. Biochemistry 40:2243–2250CrossRefGoogle Scholar
  62. 62.
    Gelpke MDS, Youngs HL, Gold MH (2000) Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants. Eur J Biochem 267:7038–7045CrossRefGoogle Scholar
  63. 63.
    Kapich AN, Korneichik TV, Hatakka A et al (2010) Oxidizability of unsaturated fatty acids and of a non-phenolic lignin structure in the manganese peroxidase-dependent lipid peroxidation system. Enzyme Microb Technol 46:136–140CrossRefGoogle Scholar
  64. 64.
    Bao WL, Fukushima Y, Jensen KA et al (1994) Oxidative degradation of non-phenolic lignin during lipid peroxidation by fungal manganese peroxidase. FEBS Lett 354:297–300CrossRefGoogle Scholar
  65. 65.
    Mester T, Ambert-Balay K, Ciofi-Baffoni S et al (2001) Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase. J Biol Chem 276:22985–22990CrossRefGoogle Scholar
  66. 66.
    Moreira PR, Almeida-Vara E, Malcata FX et al (2007) Lignin transformation by a versatile peroxidase from a novel Bjerkandera sp strain. Int Biodeterior Biodegrad 59:234–238CrossRefGoogle Scholar
  67. 67.
    Johjima T, Itoh H, Kabuto M et al (1999) Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium. Proc Natl Acad Sci USA 96:1989–1994CrossRefGoogle Scholar
  68. 68.
    Poulos TL, Edwards SL, Wariishi H et al (1993) Crystallographic refinement of lignin peroxidase at 2 Å. J Biol Chem 268:4429–4440Google Scholar
  69. 69.
    Pelletier H, Kraut J (1992) Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c. Science 258:1748–1755CrossRefGoogle Scholar
  70. 70.
    Blodig W, Smith AT, Winterhalter K et al (1999) Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase. Arch Biochem Biophys 370:86–92CrossRefGoogle Scholar
  71. 71.
    Choinowski T, Blodig W, Winterhalter K et al (1999) The crystal structure of lignin peroxidase at 1.70 Å resolution reveals a hydroxyl group on the Cb of tryptophan 171: A novel radical site formed during redox cycle. J Mol Biol 286:809–827CrossRefGoogle Scholar
  72. 72.
    Smith AT, Doyle WA, Dorlet P et al (2009) Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase. Proc Natl Acad Sci USA 106:16084–16089CrossRefGoogle Scholar
  73. 73.
    Miki Y, Tanaka H, Nakamura M et al (2006) Isolation and characterization of a novel lignin peroxidase from the white-rot basidiomycete Trametes cervina. J Fac Agr Kyushu Univ 51:99–104Google Scholar
  74. 74.
    Khindaria A, Yamazaki I, Aust SD (1996) Stabilization of the veratryl alcohol cation radical by lignin peroxidase. Biochemistry 35:6418–6424CrossRefGoogle Scholar
  75. 75.
    Tsukihara T, Honda Y, Sakai R et al (2008) Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2. Appl Environ Microbiol 74:2873–2881CrossRefGoogle Scholar
  76. 76.
    Ruiz-Dueñas FJ, Morales M, Mate MJ et al (2008) Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase. Biochemistry 47:1685–1695CrossRefGoogle Scholar
  77. 77.
    Smith AT, Doyle WA (2006) Engineered peroxidases with veratryl alcohol oxidase activity. Patent (International) WO/2006-114616Google Scholar
  78. 78.
    Khindaria A, Nie G, Aust SD (1997) Detection and characterization of the lignin peroxidase compound II- veratryl alcohol cation radical complex. Biochemistry 36:14181–14185CrossRefGoogle Scholar
  79. 79.
    Heinfling A, Ruiz-Dueñas FJ, Martínez MJ et al (1998) A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta. FEBS Lett 428:141–146CrossRefGoogle Scholar
  80. 80.
    Gelpke MDS, Lee J, Gold MH (2002) Lignin peroxidase oxidation of veratryl alcohol: Effects of the mutants H82A, Q222A, W171A, and F267L. Biochemistry 41:3498–3506CrossRefGoogle Scholar
  81. 81.
    Dawson JH, Sono M (1987) Cytochrome P-450 and chloroperoxidase – thiolate-ligated heme enzymes – spectroscopic determination of their active-site structures and mechanistic implications of thiolate ligation. Chem Rev 87:1255–1276CrossRefGoogle Scholar
  82. 82.
    Ullrich R, Nuske J, Scheibner K et al (2004) Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes. Appl Environ Microbiol 70:4575–4581CrossRefGoogle Scholar
  83. 83.
    Kluge M, Ullrich R, Dolge C et al (2009) Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation. Appl Microbiol Biotechnol 81:1071–1076CrossRefGoogle Scholar
  84. 84.
    Pecyna MJ, Ullrich R, Bittner B et al (2009) Molecular characterization of aromatic peroxygenase from Agrocybe aegerita. Appl Microbiol Biotechnol 84:885–897CrossRefGoogle Scholar
  85. 85.
    Morris DR, Hager LP (1966) Chloroperoxidase.I. Isolation and properties of crystalline glycoprotein. J Biol Chem 241:1763Google Scholar
  86. 86.
    Yi XW, Mroczko M, Manoj KM et al (1999) Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue. Proc Natl Acad Sci USA 96:12412–12417CrossRefGoogle Scholar
  87. 87.
    Yi XW, Conesa A, Punt PJ et al (2003) Examining the role of glutamic acid 183 in chloroperoxidase catalysis. J Biol Chem 278:13855–13859CrossRefGoogle Scholar
  88. 88.
    Sundaramoorthy M, Terner J, Poulos TL (1998) Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modeling studies. Chem Biol 5:461–473CrossRefGoogle Scholar
  89. 89.
    Manoj KM, Hager LP (2008) Chloroperoxidase, a Janus enzyme. Biochemistry 47:2997–3003CrossRefGoogle Scholar
  90. 90.
    Kühnel K, Blankenfeldt W, Terner J et al (2006) Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate. J Biol Chem 281:23990–23998CrossRefGoogle Scholar
  91. 91.
    Kim SJ, Ishikawa K, Hirai M et al (1995) Characteristics of a newly isolated fungus, Geotrichum candidum Dec 1, which decolorizes various dyes. J Ferment Bioeng 79:601–607CrossRefGoogle Scholar
  92. 92.
    Scheibner M, Hulsdau B, Zelena K et al (2008) Novel peroxidases of Marasmius scorodonius degrade beta-carotene. Appl Microbiol Biotechnol 77:1241–1250CrossRefGoogle Scholar
  93. 93.
    Puhse M, Szweda RT, Ma YY et al (2009) Marasmius scorodonius extracellular dimeric peroxidase – Exploring its temperature and pressure stability. BBA Proteins Proteomics 1794:1091–1098CrossRefGoogle Scholar
  94. 94.
    Zubieta C, Krishna SS, Kapoor M et al (2007) Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif. Proteins 69:223–233CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Francisco J. Ruiz-Dueñas
    • 1
  • Angel T. Martínez
    • 1
  1. 1.Centro de Investigaciones BiológicasCSICMadridSpain

Personalised recommendations