title: Insights in the kinetic mechanism of the eukaryotic Baeyer-Villiger monooxygenase BVMOAf1 from Aspergillus fumigatus Af293.
abstract: This work reports a detailed kinetic study of the recently discovered BVMOAf1 from Aspergillus fumigatus Af293. By performing steady state and pre-steady state kinetic analyses, it was demonstrated that the rate of catalysis is partially limited by the NADPH-mediated reduction of the flavin cofactor, a unique hallmark of BVMOAf1. In addition, the oxygenating C4a-(hydro)peroxyflavin intermediate could be spectrophotometrically detected and it was found to be the most stable among all analyzed BVMOs. To assess the possible influence of some residues on the kinetic features, model-inspired site-directed mutagenesis was performed. Among the mutants, the Q436A variant showed a slightly broader substrate scope and a better catalytic efficiency. In summary, this study describes for the first time the kinetic parameters for an eukaryotic BVMO.
authors.affiliation: INTEQUI-CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, CP 5700 San Luis, Argentina. Electronic address: mlmascotti@unsl.edu.ar.
authors.forename: María Laura
authors.initials: ML
authors.lastname: Mascotti
authors.affiliation: INTEQUI-CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, CP 5700 San Luis, Argentina.
authors.forename: Marcela
authors.initials: M
authors.lastname: Kurina-Sanz
authors.affiliation: IMIBIO-SL CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, CP 5700 San Luis, Argentina.
authors.forename: Maximiliano
authors.initials: M
authors.lastname: Juri Ayub
authors.affiliation: Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D005415:Flavins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D005415:Flavins; D005656:Fungal Proteins; D009249:NADP; D006899:Mixed Function Oxygenases
keywords: Aspergillus fumigatus; BVMO(Af1); Baeyer–Villiger monooxygenases; Kinetic mechanism; Site-directed mutagenesis