Journal
CHEMBIOCHEM
Volume 17, Issue 24, Pages 2312-2315Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201600484
Keywords
Baeyer-Villiger monooxygenases; cofactor specificity; enzyme catalysis; protein engineering; rational design
Funding
- German Federal State Mecklenburg-Vorpommern through state graduate scholarship (Landesgraduiertenstipendium)
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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.
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