title: How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates.
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.
authors.affiliation: Dipartimento di Chimica, Università La Sapienza and IMC-CNR Sezione Meccanismi di Reazione, Piazzale A. Moro 5, Rome, Italy, carlo.galli@uniroma1.it
authors.forename: Carlo
authors.initials: C
authors.lastname: Galli
authors.forename: Patrizia
authors.initials: P
authors.lastname: Gentili
authors.forename: Claude
authors.initials: C
authors.lastname: Jolivalt
authors.forename: Catherine
authors.initials: C
authors.lastname: Madzak
authors.forename: Raffaella
authors.initials: R
authors.lastname: Vadalà
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity; D055454:Trametes / Q000201:enzymology*; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D010636:Phenols; D042845:Laccase