Publications

• Camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions.

  Iwaki H., Grosse S., Bergeron H., Leisch H., Morley K., Hasegawa Y., Lau P.C.K.

  Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17 ... >> More

  Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 μM; kcat = 283 s(-1)) in preference to flavin adenine dinucleotide (FAD) (Km = 19 μM; kcat = 128 s(-1)). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates. << Less

  Appl. Environ. Microbiol. 79:3282-3293(2013) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

  Isupov M.N., Schroder E., Gibson R.P., Beecher J., Donadio G., Saneei V., Dcunha S.A., McGhie E.J., Sayer C., Davenport C.F., Lau P.C., Hasegawa Y., Iwaki H., Kadow M., Balke K., Bornscheuer U.T., Bourenkov G., Littlechild J.A.

  The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is en ... >> More

  The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily. << Less

  Acta Crystallogr. D 71:2344-2353(2015) [PubMed] [EuropePMC]

• 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.

  Kadow M., Loschinski K., Sass S., Schmidt M., Bornscheuer U.T.

  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-tri ... >> More

  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. << Less

  Appl. Microbiol. Biotechnol. 96:419-429(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E. coli.

  Kadow M., Balke K., Willetts A., Bornscheuer U.T., Backvall J.E.

  The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane m ... >> More

  The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor. << Less

  Appl Microbiol Biotechnol 98:3975-3986(2014) [PubMed] [EuropePMC]

• Diketocamphane enantiomer-specific 'Baeyer-Villiger' monooxygenases from camphor-grown Pseudomonas putida ATCC 17453.

  Jones K.H., Smith R.T., Trudgill P.W.

  Pseudomonas putida ATCC 17453 grew with either (+)- or (-)-camphor as sole carbon source. Enantiomer-specific 'biological Baeyer-Villiger' monooxygenases were synthesized irrespective of the camphor isomer used for growth. The two enzymes are probably the products of separate genes but showed many ... >> More

  Pseudomonas putida ATCC 17453 grew with either (+)- or (-)-camphor as sole carbon source. Enantiomer-specific 'biological Baeyer-Villiger' monooxygenases were synthesized irrespective of the camphor isomer used for growth. The two enzymes are probably the products of separate genes but showed many similarities. Each consisted of two electrophoretically identical subunits, bound flavin mononucleotide (FMN) non-covalently and accepted electrons from an induced NADH dehydrogenase which interacted with the FMN bound to the oxygenating component. They showed minor differences in M(r) with 3,6-diketocamphane 1,6-monooxygenase being the smaller enzyme. Isoelectric focussing showed the two enzymes to have different acidic pI values. Polyclonal antibodies raised against 3,6-diketocamphane 1,6-monooxygenase also cross-reacted with 2,5-diketocamphane 1,2-monooxygenase and its subunits. << Less

  J. Gen. Microbiol. 139:797-805(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.