Molecular redesign of Baeyer-Villiger Monooxygenases. Understanding and improvement of their biocatalytic properties

Baeyer-Villiger monooxygenases (BVMOs) are enzymes that catalyze the oxidation of ketones to esters. BVMOs contain a flavin cofactor (= derivative of vitamin B2) and require NADPH and oxygen for their activity. Studies have shown that these enzymes perform highly regio- and/or enantioselective oxidative reactions, which are more efficient and selective that existing chemical methods. This indicates that BVMOs can be used as biocatalysts for the synthesis of interesting fine chemicals. Phenylacetone monooxygenase (PAMO) is a thermostable BVMO that oxidizes mainly small aromatic substrates. At the beginning of this thesis the three-dimensional structure of PAMO was solved, which enabled us to identify residues that are necessary for the specific activity of the enzyme. By substituting several residues we altered PAMO to such an extent that the enzyme now catalyzes new oxidative reactions (e.g. the oxidation of indole to the dye blue indigo). By studying the catalytic mechanism of this enzyme, we also identified that an active site residue, an arginine, plays a crucial role in various reaction steps. Biotechnological application of BVMOs on a large-scale is limited due to the requirement of the expensive NADPH. To overcome this problem, we have separately fused various BVMOs to another enzyme, a phosphite dehydrogenase. This enzyme is able to regenerate NADPH at the expense of phosphite, which is a cheap substrate. These artificial bifunctional BVMOs were shown to perform efficient and selective reactions using small amounts of NADPH.