title: Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization.
abstract: The camphor-degrading Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40 years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-∆3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (−)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20 °C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k cat/K M value of 49.3 mM-1 s-1.
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Kadow
authors.forename: Kathleen
authors.initials: K
authors.lastname: Loschinski
authors.forename: Stefan
authors.initials: S
authors.lastname: Sass
authors.forename: Marlen
authors.initials: M
authors.lastname: Schmidt
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002164:Camphor / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D016958:Pseudomonas putida / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002164:Camphor; D006899:Mixed Function Oxygenases