title: Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E. coli.
abstract: The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.
authors.affiliation: Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Svante Arrhenius Väg 16C, 106 91, Stockholm, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Kadow
authors.forename: Kathleen
authors.initials: K
authors.lastname: Balke
authors.forename: Andrew
authors.initials: A
authors.lastname: Willetts
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
authors.forename: J-E
authors.initials: JE
authors.lastname: Bäckvall
mesh_terms: D002164:Camphor / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D038181:FMN Reductase / Q000235:genetics / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism*; D015870:Gene Expression; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003067:Coenzymes; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D002164:Camphor; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; D038181:FMN Reductase; C540768:Fre protein, E coli