Jump to ContentJump to Main Navigation
Show Summary Details
More options …


Open Access
See all formats and pricing
More options …

Structure and function of Aspergillus niger laccase McoG

Marta Ferraroni
  • Department of Chemistry ‘Ugo Schiff’, University of Florence, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Adrie H. Westphal
  • Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Marco Borsari
  • Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via Campi 103, I-41125 Modena, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Juan Antonio Tamayo-Ramos
  • International Research Center in Critical Raw Materials for Advanced Industrial Technologies (ICCRAM), University of Burgos, Plaza Misael Banuelos s/n, 09001, Burgos, Spain
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Prof. Dr. Fabrizio Briganti
  • Department of Chemistry ‘Ugo Schiff’, University of Florence, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Dr. Leo H. de Graaff
  • Microbial Systems Biology, Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Willem J. H. van Berkel
  • Corresponding author
  • Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, Netherlands
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2017-02-08 | DOI: https://doi.org/10.1515/boca-2017-0001


The ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2’-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid or N,N-dimethyl-p-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (ks) of wild type and variants were determined (McoG wt E°’ is +453 mV), and especially the reduced ks values of H253A and H253N show strong correlation with their low activity on phenolic compounds. In summary, our results suggest that the His253 adaptation of McoG can be beneficial for the conversion of phenolic compounds.

Keywords: Aspergillus niger; crystal structure; laccase; redox potential; metalloprotein; multicopper oxidase


  • [1] Hoegger, P.J., Kilaru, S., James, T.Y., Thacker, J.R. & Kues, U., Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences, FEBS J., 2006, 273, 2308-2326.Google Scholar

  • [2] Sakurai, T. & Kataoka, K., Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccase, Chem. Rec., 2007, 7, 220-229.CrossrefGoogle Scholar

  • [3] Quintanar, L., Stoj, C., Taylor, A.B., Hart, P.J., Kosman, D.J. & Solomon, E.I., Shall we dance? How a multicopper oxidase chooses its electron transfer partner, Acc. Chem. Res., 2007, 40, 445-452.CrossrefGoogle Scholar

  • [4] Giardina, P., Faraco, V., Pezzella, C., Piscitelli, A., Vanhulle, S. & Sannia, G., Laccases: a never-ending story, Cell. Mol. Life Sci., 2010, 67, 369-385.Google Scholar

  • [5] Baldrian, P., Fungal laccases - occurrence and properties, FEMS Microbiol. Rev., 2006, 30, 215-242.CrossrefGoogle Scholar

  • [6] Schouten, A., Wagemakers, L., Stefanato, F.L., van der Kaaij, R.M. & van Kan, J.A., Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase, Mol. Microbiol., 2002, 43, 883-894.CrossrefGoogle Scholar

  • [7] Mate, D., Garcia-Ruiz, E., Camarero, S. & Alcalde, M., Directed evolution of fungal laccases, Curr. Genomics, 2011, 12, 113-122.CrossrefGoogle Scholar

  • [8] Shraddha, Shekher, R., Sehgal, S., Kamthania, M. & Kumar, A., Laccase: microbial sources, production, purification, and potential biotechnological applications, Enzyme Res., 2011, 2011, 2011:217861.Google Scholar

  • [9] Tetsch, L., Bend, J. & Holker, U., Molecular and enzymatic characterisation of extra- and intracellular laccases from the acidophilic ascomycete Hortaea acidophila, Anton. Leeuw., 2006, 90, 183-194.Google Scholar

  • [10] Tamayo-Ramos, J.A., Barends, S., Verhaert, R.M.D. & de Graaff, L.H., The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes, Microb. Cell. Fact., 2011, 10, 78.CrossrefGoogle Scholar

  • [11] Hoshida, H., Nakao, M., Kanazawa, H., Kubo, K., Hakukawa, T., Morimasa, K., Akada, R. & Nishizawa, Y., Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts, J. Biosci. Bioeng., 2001, 92, 372-380.Google Scholar

  • [12] Cordoba Canero, D.C. & Roncero, M.I.G., Functional analyses of laccase genes from Fusarium oxysporum, Phytopathology, 2008, 98, 509-518.Google Scholar

  • [13] Courty, P.E., Hoegger, P.J., Kilaru, S., Kohler, A., Buee, M., Garbaye, J., Martin, F. & Kues, U., Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor, New Phytol., 2009, 182, 736-750.Google Scholar

  • [14] Mander, G.J., Wang, H., Bodie, E., Wagner, J., Vienken, K., Vinuesa, C., Foster, C., Leeder, A.C., Allen, G., Hamill, V., et al., Use of laccase as a novel, versatile reporter system in filamentous fungi, Appl. Environ. Microbiol., 2006, 72, 5020-5026.Google Scholar

  • [15] Tamayo-Ramos, J.A., van Berkel, W.J.H. & de Graaff, L.H., Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger, Microb. Cell. Fact., 2012, 11.Google Scholar

  • [16] Sambrook, J., Fritsch, E.F. & Maniatis, T., Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1989.Google Scholar

  • [17] de Graaff, L.H., van den Broek, H. & Visser, J., Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans, Cur. Genetics., 1988, 13, 315-321.Google Scholar

  • [18] Oliveira, J.M., van der Veen, D., de Graaff, L.H. & Qin, L., Efficient cloning system for construction of gene silencing vectors in Aspergillus niger, Appl. Microbiol. Biotechnol., 2008, 80, 917-924.Google Scholar

  • [19] Pei, J., Bong-Hyun, K., B-H. & Grishin, N.V., PROMALS3D: a tool for multiple sequence and structure alignment, Nucl. Acids Res., 2008, 36, 2295-2300.CrossrefGoogle Scholar

  • [20] Robert, X. & Gouet, P., Deciphering key features in protein structures with the new ENDscript server, Nucl. Acids Res., 2014, 42, W320-W324.CrossrefGoogle Scholar

  • [21] Pontecorvo, G., Roper, J.A., Chemmons, L.M., Macdonald, K.D. & Bufton, A.W.J., The genetics of Aspergillus nidulans, Adv. Genet., 1953, 5, 141-238.Google Scholar

  • [22] van der Veen, D., Oliveira, J.M., van den Berg, W.A.M. & de Graaff, L.H., Analysis of variance components reveals the contribution of sample processing to transcript variation, Appl. Environ. Microbiol., 2009, 75, 2414-2422.Google Scholar

  • [23] Laviron, E., General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems, J. Electroanal. Chem. Interfacial Electrochem., 1979, 101, 19-28.Google Scholar

  • [24] Yee, E.L., Cave, R.J., Guyer, K.L., Tyma, P.D. & Weaver, M.J., A survey of ligand effects upon the reaction entropies of some transition metal redox couples, J. Am. Chem. Soc., 1979, 101, 1131-1137.Google Scholar

  • [25] Yee, E.L. & Weaver, M.J., Functional dependence upon ligand composition of the reaction entropies for some transition-metal redox couples containing mixed ligands, Inorg. Chem., 1980, 19, 1077-1079.Google Scholar

  • [26] Taniguchi, V.T., Sailasuta-Scott, N., Anson, F.C. & Gray, H.B., Thermodynamics of metalloprotein electron transfer reactions, Pure Appl. Chem., 1980, 52, 2275-2281.Google Scholar

  • [27] Kabsch, W., Integration, scaling, space-group assignment and post-refinement, Acta Crystallogr. D: Biol. Crystallogr., 2010, 66(Pt 2), 133-144.Google Scholar

  • [28] Vagin, A.A. & Teplyakov, A., MOLREP: an automated program for molecular replacement, J. Appl. Cryst., 1997, 30, 1022-1025.Google Scholar

  • [29] Murshudov, G.N., Vagin, A.A. & Dodson, E.J., Refinement of macromolecular structures by the Maximum-Likelihood method, Acta Crystallogr. D: Struc. Biol., 1997, 53, 240-255.Google Scholar

  • [30] Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K., Features and development of Coot, Acta Crystallogr. D: Biol. Crystallogr., 2010, 66(Pt 4), 486-501.Google Scholar

  • [31] Lamzin, V.S., Perrakis, A. & Wilson, K.S. ARP/wARP - automated model building and refinement in International Tables for Crystallography 720-722, Kluwer Academic Publishers, Dordrecht, The Netherlands (2001).Google Scholar

  • [32] Lovell, S.C., Davis, I.W., Arendall III, W.B., de Bakker, P.I., Word, J.M., Prisant, M.G., Richardson, J.S. & Richardson, D.C., Structure validation by Calpha geometry: phi,psi and Cbeta deviation, Proteins, 2003, 15, 437-450.CrossrefGoogle Scholar

  • [33] Rodriguez-Delgado, M.M., Aleman-Nava, G.S., Rodriguez- Delgado, J.M., Dieck-Assad, G., Martinez-Chapa, S.O., Barcelo, D. & Parra, R., Laccase-based biosensors for detection of phenolic compounds, TRAC, 2015, 74, 21-45.Google Scholar

  • [34] Kallio, J.P., Auer, S., Janis, J., Andberg, M., Kruus, K., Rouvinen, J., Koivula, A. & Hakulinen, N., Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds, J. Mol. Biol., 2009, 392, 895-909.Google Scholar

  • [35] Frasconi, M., Favero, G., Boer, H., Koivula, A. & Mazzei, F., Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin, Biochim. Biophys. Acta, 2010, 1804, 899-909.Google Scholar

  • [36] Kallio, J.P., Gasparetti, C., Andberg, M., Boer, H., Koivula, A., Kruus, K., Rouvinen, J. & Hakulinen, N., Crystal structure of an ascomycete fungal laccase from Thielavia arenaria - common structural features of asco-laccases, FEBS J., 2011, 278, 2283-2295.Google Scholar

  • [37] Battistuzzi, G., Borsari, M., Sola, M. & Francia, F., Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c, Biochemistry, 1997, 36, 16247-16258.CrossrefGoogle Scholar

  • [38] Battistuzzi, G., Borsari, M., Loschi, L., Martinelli, A. & Sola, M., Thermodynamics of the alkaline transition of cytochrome c, Biochemistry, 1999, 38, 7900-7907.CrossrefGoogle Scholar

  • [39] Taniguchi, V.T., Malmstrom, B.G., Anson, F.C. & Gray, H.B., Temperature dependence of the reduction potential of blue copper in fungal laccase, Proc. Natl. Acad. Sci. USA, 1982, 79, 3387-3389.Google Scholar

  • [40] Battistuzzi, G., Borsari, M., Loschi, L., Menziani, M.C., De Rienzo, F. & Sola, M., Control of metalloprotein reduction potential: the role of electrostatic and solvation effects probed on plastocyanin mutants, Biochemistry, 2001, 40, 6422-6430.CrossrefGoogle Scholar

  • [41] Warren, J.J., Lancaster, K.L., Richards, J.H. & Gray, H.B., Innerand outer-sphere metal coordination in blue copper proteins, J. Inorg. Biochem., 2012, 115, 119-126.Google Scholar

  • [42] Hakulinen, N., Kiiskinen, L.L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. & Rouvinen, J., Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site, Nat. Struct. Biol., 2002, 9, 601-605.Google Scholar

  • [43] Ferraroni, M., Matera, I., Chernykh, A., Kolomytseva, M., Golovleva, L.A., Scozzafava, A. & Briganti, F., Reaction intermediates and redox state changes in a blue laccase from Steccherinum ochraceum observed by crystallographic high/low X-ray dose experiments, J. Inorg. Bioch., 2012, 111, 203-209.Google Scholar

  • [44] Ferraroni, M., Myasoedova, N.M., Schmatchenko, V., Leontievsky, A.A., Golovleva, L.A., Scozzafava, A. & Briganti, F., Crystal structure of a blue laccase from Lentinus tigrinus: Evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases, BMC Struct. Biol., 2007, 7, 60.CrossrefGoogle Scholar

  • [45] Garavaglia, S., Cambria, M.T., Miglio, M., Ragusa, S., Iacobazzi, V., Palmieri, F., D’Ambrosio, C., Scaloni, A. & Rizzi, M., The structure of Rigidoporus lignosus Laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair, J. Mol. Biol., 2004, 342, 1519-1531.Google Scholar

  • [46] Komori, H., Sugiyama, R., Kataoka, K., Miyazaki, K., Higuchi, Y. & Sakurai, T., New insights into the catalytic active-site structure of multicopper oxidases, Acta Crystallogr. D: Biol. Crystallogr., 2014, D70, 772-779.CrossrefGoogle Scholar

  • [47] Bento, I., Martins, L.O., Gato Lopes, G., Armenia Carrondo, M. & Lindley, P.F., Dioxygen reduction by multi-copper oxidases; a structural perspective, Dalton Trans., 2005, 21, 3507-3513.CrossrefGoogle Scholar

  • [48] Durao, P., Bento, I., Fernandes, A.T., Melo, E.P., Lindley, P.F. & Martins, L.O., Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies, J. Biol. Inorg. Chem., 2006, 11, 514-526.Google Scholar

  • [49] Andberg, M., Hakulinen, N., Auer, S., Saloheimo, M., Koivula, A., Rouvinen, J. & Kruus, K., Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure, FEBS J., 2009, 276, 6285-6300.Google Scholar

  • [50] Zumarraga, M., Camarero, S., Shleev, S., Martinez-Arias, A., Ballesteros, A., Plou, F.J. & Alcalde, M., Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra, Proteins, 2008, 71, 250-260.Google Scholar

  • [51] Bertrand, T., Jolivalt, C., Briozzo, P., Caminade, E., Joly, N., Madzak, C. & Mougin, C., Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics, Biochemistry, 2002, 41, 7325-7333.CrossrefGoogle Scholar

  • [52] Matera, I., Gullotto, A., Tilli, I., Ferraroni, M., Scozzafava, A. & Briganti, F., Crystal structure of the blue multicopper oxidase from the white-rot fungus Trametes trogii complexed with p-toluate, Inorg. Chim. Acta, 2008, 361, 4129-4137.Google Scholar

  • [53] Enguita, F. J., Marcal, D., Martins, L.O., Grenha, R., Henriques, A.O., Lindley, P.F., Carrondo, M.A., Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis. J. Biol. Chem., 2004, 279, 23472-23476.Google Scholar

  • [54] Roberts, S.A., Wildner, G.F., Grass, G., Weichsel, A., Ambrus, A., Rensing, C. & Montfort, W.R., A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO, J. Biol. Chem., 2003, 278, 31958-31963.Google Scholar

About the article

Received: 2016-10-10

Accepted: 2016-12-08

Published Online: 2017-02-08

Published in Print: 2017-01-01

Citation Information: Biocatalysis, Volume 3, Issue 1, Pages 1–21, ISSN (Online) 2353-1746, DOI: https://doi.org/10.1515/boca-2017-0001.

Export Citation

© 2017. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Majid Haddad Momeni, Paolo Bollella, Roberto Ortiz, Esben Thormann, Lo Gorton, and Maher Abou Hachem
BMC Biotechnology, 2019, Volume 19, Number 1
Bart van Beusekom, Thomas Lütteke, and Robbie P. Joosten
Acta Crystallographica Section F Structural Biology Communications, 2018, Volume 74, Number 8, Page 463

Comments (0)

Please log in or register to comment.
Log in