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.
abstract: 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.
authors.affiliation: Department of Life Science & Biotechnology and ORDIST, Kansai University, Osaka, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Iwaki
authors.forename: Stephan
authors.initials: S
authors.lastname: Grosse
authors.forename: Hélène
authors.initials: H
authors.lastname: Bergeron
authors.forename: Hannes
authors.initials: H
authors.lastname: Leisch
authors.forename: Krista
authors.initials: K
authors.lastname: Morley
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D002164:Camphor / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D038181:FMN Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics
chemical_list: D005182:Flavin-Adenine Dinucleotide; D000105:Acetyl Coenzyme A; D002164:Camphor; D005486:Flavin Mononucleotide; D010105:Oxygenases; C115882:3,6-diketocamphane monooxygenase; C047193:2,5-diketocamphane 1,2-monooxygenase; D038181:FMN Reductase