@article{oai:kansai-u.repo.nii.ac.jp:00000689,
 author = {Iwaki, Hiroaki and Grosse, Stephan and Bergeron, Helene and Leisch, Hannes and Morley, Krista and Hasegawa, Yoshie and Lau, Peter C.K},
 journal = {Applied and Environmental Microbiology},
 month = {Mar},
 note = {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 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 is hitherto ill-defined. In this study, a 37-kDa Fred was purified from 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 mass of 18-kDa that uses NADH as electron donor (Km = 32 μM) and it catalyzes the reduction of FMN (Km = 3.6 μM; kcat = 283 s-1) in preference to FAD (Km = 19 μM; kcat = 128 s-1). Sequence determination of ∼40-kb of the camphor (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 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.},
 title = {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},
 year = {2013}
}