title: Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase.
abstract: Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Andy
authors.initials: A
authors.lastname: Beier
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Bordewick
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Maika
authors.initials: M
authors.lastname: Genz
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Sandy
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA, Nijmegen, The Netherlands.
authors.forename: Tom
authors.initials: T
authors.lastname: van den Bergh
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Peters
authors.affiliation: Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA, Nijmegen, The Netherlands.
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Joosten
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Uwe T
authors.identifier: http://orcid.org/0000-0003-0685-2696
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000150:Acinetobacter / Q000201:enzymology; D001665:Binding Sites; D055162:Biocatalysis; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C019961:cyclohexanone oxygenase
keywords: Baeyer-Villiger monooxygenases; cofactor specificity; enzyme catalysis; protein engineering; rational design