Affiliation:
1. Department of Life Science & Biotechnology and ORDIST, Kansai University, Osaka, Japan
2. National Research Council Canada, Montreal, Quebec, Canada
3. Departments of Chemistry and Microbiology & Immunology, McGill University, Montreal, Quebec, Canada
4. FQRNT Centre in Green Chemistry and Catalysis, Montreal, Quebec, Canada
Abstract
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 (
K
m
= 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (
K
m
= 3.6 μM;
k
cat
= 283 s
−1
) in preference to flavin adenine dinucleotide (FAD) (
K
m
= 19 μM;
k
cat
= 128 s
−1
). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (
camE
25–1
for 2,5-DKCMO-1 and
camE
25–2
for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (
camE
36
). 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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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