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

Download
  1. Get@NRC: 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 (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1128/AEM.03958-12
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleApplied and Environmental Microbiology
ISSN0099-2240
Volume79
Issue10
Pages32823293; # of pages: 12
SubjectBaeyer Villiger oxidation; Baeyer-Villiger reaction; Biochemical properties; Flavin adenine dinucleotide; Flavin mono nucleotides (FMN); Pulsed field gel electrophoresis; Recombinant expression; Recombinant plasmid; Cams; DNA; Electrophoresis; Escherichia coli; Genes; Genetic engineering; Ketones; Oxygenation; Substrates; Camphor; bacteriology; bacterium; catalysis; electrokinesis; enzyme activity; gene expression; ketone; oxidation; plasmid; recombination; terpene
AbstractWhereas 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.
Publication date
LanguageEnglish
AffiliationAquatic and Crop Resource Development; National Research Council Canada
Peer reviewedYes
NPARC number21270361
Export citationExport as RIS
Report a correctionReport a correction
Record identifier13e011cb-87a2-4755-b6f4-95f98f5c7b9a
Record created2014-02-05
Record modified2016-05-09
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)