title: Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution.
abstract: There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2 O2 , this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, C/Marie Curie n° 2L10, 28049, Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Molina-Espeja
authors.affiliation: Anaxomics Biotech, Balmes 89, 08008, Barcelona, Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: Cañellas
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, C/Marie Curie n° 2L10, 28049, Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.affiliation: TU Dresden, International Institute Zittau, Department of Bio- and Environmental Sciences, Markt 23, 02763, Zittau, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hofrichter
authors.affiliation: Anaxomics Biotech, Balmes 89, 08008, Barcelona, Spain.|Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, 08034, Barcelona, Spain.
authors.forename: Fatima
authors.initials: F
authors.lastname: Lucas
authors.affiliation: Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, 08034, Barcelona, Spain.|ICREA: Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, 08010, Barcelona, Spain.
authors.forename: Victor
authors.initials: V
authors.lastname: Guallar
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, C/Marie Curie n° 2L10, 28049, Madrid, Spain. malcalde@icp.csic.es.
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
mesh_terms: D055362:Agrocybe / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009281:Naphthalenes / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism*; D017354:Point Mutation; D015202:Protein Engineering
chemical_list: D009281:Naphthalenes; D009284:Naphthols; C031721:naphthalene; C029350:1-naphthol; D006899:Mixed Function Oxygenases; C022333:peroxygenase
keywords: 1-naphthol; directed evolution; enzyme catalysis; peroxidative activity; peroxygenases