Studying the binding mechanisms of veratryl alcohol to P. chrysosporium lignin peroxidase: insights from theoretical approaches

  • Rodrigo Recabarren
  • Isabel Fuenzalida-Valdivia
  • Jans Alzate-MoralesEmail author
Regular Article
Part of the following topical collections:
  1. CHITEL 2015 - Torino - Italy


Veratryl alcohol (VA) is the main substrate of lignin peroxidase (LiP), a key lignin-degrading enzyme. A redox mediator role, in the lignin degradation process, has been attributed to this molecule; however, many unanswered questions remain about its action mechanism. In this investigation, the basic aspects of a plausible action mechanism, this means VA binding modes to Phanerochaete chrysosporium LiP, were addressed. Docking calculations were used to obtain LiP–VA complexes near to Trp171, the active redox residue where VA is oxidized. Our results show that VA interacts at Trp171 helped by hydrogen bonding interactions with the acidic amino acids Asp264 and Glu168, as well as by hydrophobic interactions with Phe267, confirming previous experimental findings. MM–GBSA calculations, molecular dynamics simulations, and cluster analysis gave further insights into the energetic preferences of the different binding modes and the stability of LiP–VA complexes. A hydrophobic concave ditch, next to Trp171, was observed to stabilize VA at LiP surface, confirming previous suggestions based on the LiP crystal structure. A detailed analysis of the interactions in this site is provided. These findings are expected to be the basis for site-directed mutagenesis and QM/MM experiments that will prove the importance of the identified residues.


Lignin-degrading enzymes LiP Biotransformations Molecular dynamics MM/GBSA Value-added products 



J.H.A.M. and I.F.V. acknowledge the financial support from project FONDECYT No. 1140618. R.R. acknowledges support from a doctoral fellowship CONICYT-PCHA/Folio 21130949. We thank Prof. Angel Martínez, from Centro de Investigaciones Biológicas (CIB), Madrid, Spain, for his help in the revision and valuable comments on the first draft of the manuscript. 

Supplementary material

214_2016_1828_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1232 kb)


  1. 1.
    Heitner C, Dimmel D, Schmidt J (2011) Lignin and lignans: advances in chemistry. CRC Press, Boca Raton, FLGoogle Scholar
  2. 2.
    Martínez ÁT et al (2010) Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. Int Microbiol 8:195–204Google Scholar
  3. 3.
    Wong DWS (2009) Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 157:174–209CrossRefGoogle Scholar
  4. 4.
    Kirk TK, Farrell RL (1987) Enzymatic ‘combustion’: the microbial degradation of lignin. Annu Rev Microbiol 41:465–501CrossRefGoogle Scholar
  5. 5.
    Eriksson K-EL, Blanchette RA, Ander P (1990) Microbial and enzymatic degradation of wood and wood components. Springer, BerlinCrossRefGoogle Scholar
  6. 6.
    Kersten P, Cullen D (2007) Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium. Fungal Genet Biol 44:77–87CrossRefGoogle Scholar
  7. 7.
    Tien M, Kirk TK (1983) Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium burds. Science 221:661–663CrossRefGoogle Scholar
  8. 8.
    Hammel KE et al (1993) Ligninolysis by a purified lignin peroxidase. J Biol Chem 268:12274–12281Google Scholar
  9. 9.
    Valli K, Wariishi H, Gold MH (1990) Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation. Biochemistry 29:8535–8539CrossRefGoogle Scholar
  10. 10.
    Banci L, Ciofi-Baffoni S, Tien M (1999) Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers. Biochemistry 38:3205–3210CrossRefGoogle Scholar
  11. 11.
    Glenn JK, Morgan MA, Mayfield MB, Kuwahara M, Gold MH (1983) An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium. Biochem Biophys Res Commun 114:1077–1083CrossRefGoogle Scholar
  12. 12.
    Chance B (1949) The reaction of catalase and cyanide. J Biol Chem 179:1299–1309Google Scholar
  13. 13.
    Niladevi KN (2009) In: Nigam PS, Pandey A (eds) Biotechnology for agro-industrial residues utilisation. Springer, Dordrecht, pp 397–414CrossRefGoogle Scholar
  14. 14.
    Novotný Č et al (2004) Ligninolytic fungi in bioremediation: extracellular enzyme production and degradation rate. Soil Biol Biochem 36:1545–1551CrossRefGoogle Scholar
  15. 15.
    Durán N, Esposito E (2000) Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater and soil treatment: a review. Appl Catal B Environ 28:83–99CrossRefGoogle Scholar
  16. 16.
    Regalado C, García-Almendárez BE, Duarte-Vázquez MA (2004) Biotechnological applications of peroxidases. Phytochem Rev 3:243–256CrossRefGoogle Scholar
  17. 17.
    Choinowski T, Blodig W, Winterhalter KH, Piontek K (1999) The crystal structure of lignin peroxidase at 1.70 Å resolution reveals a hydroxy group on the Cβ of tryptophan 171: a novel radical site formed during the redox cycle1. J Mol Biol 286:809–827CrossRefGoogle Scholar
  18. 18.
    Piontek K, Glumoff T, Winterhalter K (1993) Low pH crystal structure of glycosylated lignin peroxidase from Phanerochaete chrysosporium at 2.5 Å resolution. FEBS Lett 315:119–124CrossRefGoogle Scholar
  19. 19.
    Edwards SL, Raag R, Wariishi H, Gold MH, Poulos TL (1993) Crystal structure of lignin peroxidase. Proc Natl Acad Sci USA 90:750–754CrossRefGoogle Scholar
  20. 20.
    Poulos TL, Edwards SL, Wariishi H, Gold MH (1993) Crystallographic refinement of lignin peroxidase at 2 A. J Biol Chem 268:4429–4440Google Scholar
  21. 21.
    Schoemaker HE, Lundell TK, Hatakka AI, Piontek K (1994) The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: the redox cycle revisited. FEMS Microbiol Rev 13:321–331CrossRefGoogle Scholar
  22. 22.
    Lundquist K, Kirk TK (1978) De novo synthesis and decomposition of veratryl alcohol by a lignindegrading basidiomycete. Phytochemistry 17:1676CrossRefGoogle Scholar
  23. 23.
    Francesca Gerini M, Roccatano D, Baciocchi E, Nola AD (2003) Molecular dynamics simulations of lignin peroxidase in solution. Biophys J 84:3883–3893CrossRefGoogle Scholar
  24. 24.
    Blodig W et al (1998) Autocatalytic formation of a hydroxy group at C beta of Trp171 in lignin peroxidase. Biochemistry 37:8832–8838CrossRefGoogle Scholar
  25. 25.
    Doyle WA, Blodig W, Veitch NC, Piontek K, Smith AT (1998) Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis. Biochemistry 37:15097–15105CrossRefGoogle Scholar
  26. 26.
    Timofeevski SL, Nie G, Reading NS, Aust SD (1999) Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis. Biochem Biophys Res Commun 256:500–504CrossRefGoogle Scholar
  27. 27.
    Sollewijn-Gelpke MD, 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
  28. 28.
    Mester T et al (2001) Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase. J Biol Chem 276:22985–22990CrossRefGoogle Scholar
  29. 29.
    Harvey PJ, Schoemaker HE, Palmer JM (1986) Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium. FEBS Lett 195:242–246CrossRefGoogle Scholar
  30. 30.
    Goodwin DC, Aust SD, Grover TA (1995) Evidence for veratryl alcohol as a redox mediator in lignin peroxidase-catalyzed oxidation. Biochemistry 34:5060–5065CrossRefGoogle Scholar
  31. 31.
    Candeias LP, Harvey PJ (1995) Lifetime and reactivity of the veratryl alcohol radical cation. Implications for lignin peroxidase catalysis. J Biol Chem 270:16745–16748CrossRefGoogle Scholar
  32. 32.
    Baciocchi E, Bietti M, Francesca Gerini M, Lanzalunga O (2002) The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation. Biochem Biophys Res Commun 293:832–835CrossRefGoogle Scholar
  33. 33.
    Khindaria A, Yamazaki I, Aust SD (1996) Stabilization of the veratryl alcohol cation radical by lignin peroxidase. Biochemistry 35:6418–6424CrossRefGoogle Scholar
  34. 34.
    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
  35. 35.
    Johjima T et al (1999) Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium. Proc Natl Acad Sci USA 96:1989–1994CrossRefGoogle Scholar
  36. 36.
    Baciocchi E, Fabbri C, Lanzalunga O (2003) Lignin peroxidase-catalyzed oxidation of nonphenolic trimeric lignin model compounds: fragmentation reactions in the intermediate radical cations. J Org Chem 68:9061–9069CrossRefGoogle Scholar
  37. 37.
    Hammel KE, Moen MA (1991) Depolymerization of a synthetic lignin in vitro by lignin peroxidase. Enzyme Microb Technol 13:15–18CrossRefGoogle Scholar
  38. 38.
    Paszczynski A, Crawford RL (1991) Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol. Biochem Biophys Res Commun 178:1056–1063CrossRefGoogle Scholar
  39. 39.
    Chung N, Aust SD (1995) Veratryl alcohol-mediated indirect oxidation of phenol by lignin peroxidase. Arch Biochem Biophys 316:733–737CrossRefGoogle Scholar
  40. 40.
    Huang X et al (2003) The roles of veratryl alcohol and nonionic surfactant in the oxidation of phenolic compounds by lignin peroxidase. Biochem Biophys Res Commun 311:491–494CrossRefGoogle Scholar
  41. 41.
    Smith AT, Doyle WA (2006) Engineered peroxidases with veratryl alcohol oxidase activity. International Patent. WO/2006-114616, 2 Nov 2006Google Scholar
  42. 42.
    Chen M et al (2011) Understanding lignin-degrading reactions of ligninolytic enzymes: binding affinity and interactional profile. PLoS One 6:e25647CrossRefGoogle Scholar
  43. 43.
    Miki Y et al (2013) Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism. Biochem J 452:575–584CrossRefGoogle Scholar
  44. 44.
    Miki Y, Ichinose H, Wariishi H (2010) Molecular characterization of lignin peroxidase from the white-rot basidiomycete Trametes cervina: a novel fungal peroxidase. FEMS Microbiol Lett 304:39–46CrossRefGoogle Scholar
  45. 45.
    Miki Y et al (2011) Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine. J Biol Chem 286:15525–15534CrossRefGoogle Scholar
  46. 46.
    Smith AT, Doyle WA, Dorlet P, Ivancich A (2009) Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase. Proc Natl Acad Sci 106:16084–16089CrossRefGoogle Scholar
  47. 47.
    Bernini C, Pogni R, Basosi R, Sinicropi A (2012) The nature of tryptophan radicals involved in the long-range electron transfer of lignin peroxidase and lignin peroxidase-like systems: insights from quantum mechanical/molecular mechanics simulations. Proteins Struct Funct Bioinform 80:1476–1483CrossRefGoogle Scholar
  48. 48.
    Piontek K, Smith AT, Blodig W (2001) Lignin peroxidase structure and function. Biochem Soc Trans 29:111–116CrossRefGoogle Scholar
  49. 49.
    Berman HM et al (2000) The protein data bank. Nucleic Acids Res 28:235–242CrossRefGoogle Scholar
  50. 50.
    Maestro (2014) Version 9.7, Schrödinger, LLC, New York, NYGoogle Scholar
  51. 51.
    Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman W (2013) Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 27:221–234CrossRefGoogle Scholar
  52. 52.
    Schrödinger Suite 2014-1 Protein Preparation WizardGoogle Scholar
  53. 53.
    Blodig W, Smith AT, Doyle WA, Piontek K (2001) Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171. Implications for the reaction mechanism. J Mol Biol 305:851–861CrossRefGoogle Scholar
  54. 54.
    Olsson MHM, Søndergaard CR, Rostkowski M, Jensen JH (2011) PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J Chem Theory Comput 7:525–537CrossRefGoogle Scholar
  55. 55.
    Glide (2014) Version 6.2, Schrödinger, LLC, New York, NYGoogle Scholar
  56. 56.
    Friesner RA et al (2004) Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47:1739–1749CrossRefGoogle Scholar
  57. 57.
    Halgren TA et al (2004) Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem 47:1750–1759CrossRefGoogle Scholar
  58. 58.
    Banks JL et al (2005) Integrated modeling program, applied chemical theory (IMPACT). J Comput Chem 26:1752–1780CrossRefGoogle Scholar
  59. 59.
    Desmond Molecular Dynamics System (2013) Version 3.4, D. E. Shaw Research, New York, NYGoogle Scholar
  60. 60.
    Berendsen HJC, Postma JPM, van Gunsteren WF, Hermans J (1981) In: Pullman B (ed) Intermolecular forces. Springer, Dordrecht, pp 331–342CrossRefGoogle Scholar
  61. 61.
    Nosé S (1984) A unified formulation of the constant temperature molecular dynamics methods. J Chem Phys 81:511–519CrossRefGoogle Scholar
  62. 62.
    Martyna GJ, Tobias DJ, Klein ML (1994) Constant pressure molecular dynamics algorithms. J Chem Phys 101:4177–4189CrossRefGoogle Scholar
  63. 63.
    Essmann U et al (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593CrossRefGoogle Scholar
  64. 64.
    Tuckerman M, Berne BJ, Martyna GJ (1992) Reversible multiple time scale molecular dynamics. J Chem Phys 97:1990–2001CrossRefGoogle Scholar
  65. 65.
    Prime (2014) Version 3.5, Schrödinger, LLC, New York, NYGoogle Scholar
  66. 66.
    Jacobson MP et al (2004) A hierarchical approach to all-atom protein loop prediction. Proteins Struct Funct Bioinform 55:351–367CrossRefGoogle Scholar
  67. 67.
    Li J et al (2011) The VSGB 2.0 model: a next generation energy model for high resolution protein structure modeling. Proteins Struct Funct Bioinform 79:2794–2812CrossRefGoogle Scholar
  68. 68.
    Mulakala C, Viswanadhan VN (2013) Could MM–GBSA be accurate enough for calculation of absolute protein/ligand binding free energies? J Mol Graph Model 46:41–51CrossRefGoogle Scholar
  69. 69.
    Greenidge PA, Kramer C, Mozziconacci J-C, Wolf RM (2013) MM/GBSA binding energy prediction on the PDBbind data set: successes, failures, and directions for further improvement. J Chem Inf Model 53:201–209CrossRefGoogle Scholar
  70. 70.
    MacroModel (2014) Version 10.3, Schrödinger, LLC, New York, NYGoogle Scholar
  71. 71.
    Shao J, Tanner SW, Thompson N, Cheatham TE (2007) Clustering molecular dynamics trajectories: 1. Characterizing the performance of different clustering algorithms. J Chem Theory Comput 3:2312–2334CrossRefGoogle Scholar
  72. 72.
    Lama D et al (2013) Rational optimization of conformational effects induced by hydrocarbon staples in peptides and their binding interfaces. Sci Rep 3:3451. doi: 10.1038/srep03451 Google Scholar
  73. 73.
    Hayes JM et al (2011) Kinetics, in silico docking, molecular dynamics, and MM–GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: the role of water molecules examined. Proteins 79:703–719CrossRefGoogle Scholar
  74. 74.
    Takashima S et al (2007) Correlation between cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea cellobiohydrolase 1. FEBS Lett 581:5891–5896CrossRefGoogle Scholar
  75. 75.
    Linder M et al (1995) Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I. Protein Sci 4:1056–1064CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Rodrigo Recabarren
    • 1
  • Isabel Fuenzalida-Valdivia
    • 1
  • Jans Alzate-Morales
    • 1
    Email author
  1. 1.Centro de Bioinformática y Simulación Molecular, Facultad de IngenieríaUniversidad de TalcaTalcaChile

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