Research Article: Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007

Date Published: June 23, 2011

Publisher: Springer

Author(s): Maria Kadow, Stefan Saß, Marlen Schmidt, Uwe T Bornscheuer.


Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.

Partial Text

The discovery of the enzymatic Baeyer-Villiger reaction is closely connected to the exploration of the biodegradation of camphor (1) in Pseudomonads (Figure 1). Initial studies on the microbial decomposition of (+)-1 by Pseudomonas putida NCIMB 10007 isolated from sewage sludge were already carried out in 1959 (Bradshaw et al. 1959) and the involved enzymes were separated and characterized during the following decade. In studies of the enzymatic lactonization of the intermediate 2,5-diketocamphane (3) from the (+)-camphor-grown organism it was shown that two enzyme fractions were responsible for the Baeyer-Villiger-monooxygenase (BVMO) catalyzed reaction step (Conrad et al. 1961). The first enzyme turned out to be a FMN-coupled NADH-dehydrogenase [EC], while the second subunit was claimed to be a ketolactonase. Since mechanistic similarities to the chemical Baeyer-Villiger oxidation of bicyclic ketones (Meinwald and Frauenglass 1960) were detected, the nomenclature of the ketolactonase was changed to a BVMO. In 1965 a second lactonizing system for the degradation of (-)-1 was found (Conrad et al. 1965a). Thus it was claimed that (+)-1 and its derivatives were only converted by the (+)-camphor induced 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO), while (-)-1 is converted by the (-)-camphor induced 3,6-diketocamphane 1,6-monooxygenase (Jones et al. 1993) (Figure 1). Later it was claimed, that whichever enantiomer of camphor is given to the growth medium, both diketocamphane monooxygenases are induced (Gagnon et al. 1994). The ability to decompose camphor turned out to be inducible in several fluorescent Pseudomonads, where most of the involved enzymes, including both type II monooxygenases, are located on a 230 kb (165 MDa) plasmid (CAM plasmid, Figure 2) (Chakrabarty 1976).

The oxygenating subunit of the 2,5-diketocamphane monooxygenase was successfully cloned and overexpressed recombinantly in E. coli as the heterologous expression host. Hence, this enzyme is now easy available at stable quality and protein engineering studies are possible for the first time. The purification using the N-terminal His-tag via nickel based affinity chromatography turned out to be efficient and fast. While in previous purifications of the enzyme from wild type cultivations, huge culture volumes were used, in this study drastically smaller amounts of heterologous culture is needed to produce comparable amounts of pure protein. Conrad et al. used a 10 L culture and obtained 240 mL crude extract to produce 52 mg of pure protein via a chromatography based purification protocol with three steps, which corresponds to a recovery of 15% (Conrad et al. 1965a). 28 years later Jones et al. were able to increase the purity and the yield up to 19.5%. From a 10 L culture volume 49 mg of pure enzyme were obtained (Jones et al. 1993). In this work 8 mg of pure protein were achieved out of a 400 mL culture, which highlights the advantages of recombinant expression and the fusion of an enzyme to a His-tag.

FMN: flavin mononucleotide; NADH: nicotinamide adenine dinucleotide; BVMO: Baeyer-Villiger monooxygenase; 2,5-DKCMO: 2,5-diketocamphane 1,2-monooxygenase

The authors declare that they have no competing interests.




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