title: Extending the substrate scope of a Baeyer-Villiger monooxygenase by multiple-site mutagenesis.
abstract: Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer-Villiger monooxygenases with new specificities.
authors.affiliation: Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors.forename: Hanna M
authors.initials: HM
authors.lastname: Dudek
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Fink
authors.forename: Amol V
authors.initials: AV
authors.lastname: Shivange
authors.forename: Alexander
authors.initials: A
authors.lastname: Dennig
authors.forename: Marko D
authors.initials: MD
authors.lastname: Mihovilovic
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D000096:Acetone / Q000031:analogs & derivatives / Q000378:metabolism; D005820:Genetic Testing; D007659:Ketones / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D013379:Substrate Specificity
chemical_list: D007659:Ketones; D000096:Acetone; D006899:Mixed Function Oxygenases; C008863:1-phenyl-2-propanone