1. Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase
- Author
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Sven Bordewick, Tom van den Bergh, Henk-Jan Joosten, Uwe T. Bornscheuer, Sandy Schmidt, Christin Peters, Maika Genz, and Andy Beier
- Subjects
0301 basic medicine ,Stereochemistry ,Cyclohexanone ,Sequence alignment ,Molecular Dynamics Simulation ,Protein Engineering ,01 natural sciences ,Biochemistry ,Cofactor ,Enzyme catalysis ,Mixed Function Oxygenases ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Molecule ,Molecular Biology ,Binding Sites ,biology ,Acinetobacter ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Rational design ,Protein engineering ,Monooxygenase ,Recombinant Proteins ,0104 chemical sciences ,Protein Structure, Tertiary ,Kinetics ,030104 developmental biology ,biology.protein ,Biocatalysis ,Mutagenesis, Site-Directed ,Oxygenases ,Molecular Medicine ,NADP - 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.
- Published
- 2016