Abstract
In spite of its broad specificity among phenols, Trametes versicolor laccase hardly succeeds in oxidizing hindered substrates. To improve the oxidation ability of this laccase towards bulky phenolic substrates, we designed a series of single-point mutants on the basis of the amino-acid layout inside the reducing substrate active site known from the crystal structure of the enzyme. Site-directed mutagenesis has addressed four phenylalanine residues in key positions 162, 265, 332, and 337 at the entrance of the binding pocket, as these residues appeared instrumental for docking of the substrate. These phenylalanines were replaced by smaller-sized but still apolar alanines. A double mutant F162A/F332A was also designed. Measurement of the oxidation efficiency towards encumbered phenols has shown that mutant F162A was more efficient than the wild-type laccase. The double mutant F162A/F332A led to 98% consumption of bisphenol A in only 5 h and was more efficient than the single mutants in the aerobic oxidation of this bulky substrate. In contrast, lack of appropriate hydrophobic interactions with the substrate possibly depresses the oxidation outcome with mutants F265A and F332A. One explanation for the lack of reactivity of mutant F337A, supported by literature reports, is that this residue is part of the second coordination shell of T1 Cu. A mutation at this position thus leads to a drastic coordination shell destabilization. Thermal stability of the mutants and their resistance in a mixed water–dioxane solvent have also been investigated.
Similar content being viewed by others
References
Aweke Tadesse M, D’Annibale A, Galli C, Gentili P, Sergi F (2008) An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates. Org Biomol Chem 6:868–878
Baldrian P (2006) Fungal laccases—occurrence and properties. FEMS Microbiol Rev 30:215–242
Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C (2002) Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry 41:7325–7333
Camarero S, Ibarra D, Martínez AT (2007) Paper pulp delignification using laccase and natural mediators. Enzyme Microbiol Technol 40:1264–1271
Choinowski T, Antorini M, Piontek K (2002) Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-Ǻ resolution containing a full complement of coppers. J Biol Chem 277:37663–37669
d’Acunzo F, Galli C, Gentili P, Sergi F (2006) Mechanistic and steric issues in the oxidation of phenolic and non-phenolic compounds by laccase or laccase-mediator systems. The case of bifunctional substrates. New J Chem 30:583–591
d'Acunzo F, Barreca AM, Galli C (2004) Determination of the activity of laccase, and mediated oxidation of a lignin model compound, in aqueous–organic mixed solvents. J Mol Catal B Enzym 31:25–30
DeLano WL (2002) PyMol. DeLano Scientific, San Carlos
Enguita FJ, Marçal D, Martins LO, Grenha R, Henriques AO, Lindley PF, Carrondo MA (2004) Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis. J Biol Chem 279:23472–23476
Gianfreda L, Xu F, Bollag J-M (1999) Laccases: a useful group of oxidoreductive enzymes. Bioremediat J 3:1–25
Giardina P, Faraco V, Pezzella C, Piscitelli A, Vanhulle S, Sannia G (2010) Laccases: a never-ending story. Cell Mol Life Sci 67:369–385
Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C (2005) Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol 66:450–456
Jönsson L, Sjöström K, Häggstrom I, Nyman PO (1995) Characterization of a laccase gene from the white-rot fungus Trametes versicolor and structural features of basidiomycete laccases. Biochim Biophys Acta 1251:210–215
Kallio JP, Hakulinen N, Rouvinen J (2009) Structure function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds. J Mol Biol 392:895–909
Koschorreck K, Richter SM, Swierczek A, Beifuss U, Schmid RD, Urlacher VB (2008) Comparative characterization of four laccases from Trametes versicolor concerning phenolic C–C coupling and oxidation of PAHs. Arch Biochem Biophys 474:213–219
Le Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44
Madzak C, Gaillardin C, Beckerich J-M (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109:63–81
Madzak C, Mimmi MC, Caminade E, Brault A, Baumberger S, Briozzo P, Mougin C, Jolivalt C (2006) Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis. Protein Eng Des Sel 19:77–84
Matera I, Gullotto A, Tilli S, Ferraroni M, Scozzafava A, Briganti F (2008) Crystal structure of the blue multicopper oxidase from the white-rot fungus Trametes trogii complexed with p-toluate. Inorg Chim Acta 361:4129–4137
Messerschmidt A (1997) Multi-copper oxidases. World Scientific, Singapore
Mohamad SB, Ong AL, Ripen AM (2008) Evolutionary trace analysis at the ligand binding site of laccase. Bioinformation 2:369–372
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612
Rodríguez Couto S, Toca Herrera JL (2006) Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 24:500–513
Sivropoulou A, Papanikolaou E, Nikolaou C, Kokkini S, Lanaras T, Arsenakis M (1996) Antimicrobial and cytotoxic activities of origanum essential oils. J Agr Food Chem 44:1202–1205
Solomon EI, Sundaram UM, Machonkin TE (1996) Multicopper oxidases and oxygenases. Chem Rev 96:2563–2606
Suatoni JC, Snyder RE, Clark RO (1961) Voltammetric studies of phenol and aniline ring substitution. Anal Chem 33:1894–1897
Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26
Widsten P, Kandelbauer A (2008) Laccase applications in the forest products industry: a review. Enzyme Microbiol Technol 42:293–307
Xu F (1996) Oxidation of phenols, anilines, and benzenethiols by fungal laccases: correlation between activity and redox potentials as well as halide inhibition. Biochemistry 35:7608–7614
Xu F, Shin W, Brown SH, Wahleithner JA, Sundaram UM, Solomon EI (1996) A study of a series of recombinant fungal laccases and birilubin oxidase that exhibit significant differences in redox potential, substrate specificity and stability. Biochim Biophys Acta 1292:303–311
Yanagisawa S, Crowley PB, Firbank SJ, Lawler AT, Hunter DM, McFarlane W, Li C, Kohzuma T, Banfield MJ, Dennison C (2008) π-Interaction tuning of the active site properties of metalloproteins. J Am Chem Soc 130:15420–15428
Yaver DS, Xu F, Golightly EJ, Brown KM, Brown SH, Rey MW, Schneider P, Halkier T, Mondorf K, Dalbøge H (1996) Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa. Appl Environ Microbiol 62:834–841
Acknowledgments
This work was supported by the Italian MIUR (COFIN and FIRB grants), by grants from the University of Roma ‘La Sapienza’ and by a fellowship to R. Vadalà. The doctorate thesis of R. Vadalà was run in collaboration between the University of Rome ‘La Sapienza’ and the Ecole Nationale Supérieure de Chimie de Paris (C. Jolivalt).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Galli, C., Gentili, P., Jolivalt, C. et al. How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates. Appl Microbiol Biotechnol 91, 123–131 (2011). https://doi.org/10.1007/s00253-011-3240-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-011-3240-4