Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Kadow M

PROVIDER: S-EPMC3222318 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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

Kadow Maria M Saß Stefan S Schmidt Marlen M Bornscheuer Uwe T UT

AMB Express 20110623 1

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 ...[more]

PMID: 21906366

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Project description:Researching the involvement of molecular oxygen in the degradation of the naturally occurring bicyclic terpene camphor has generated a six-decade history of fascinating monooxygenase biochemistry. While an extensive bibliography exists reporting the many varied studies on camphor 5-monooxygenase, the initiating enzyme of the relevant catabolic pathway in Pseudomonas putida ATCC 17453, the equivalent recorded history of the isoenzymic diketocamphane monooxygenases, the enzymes that facilitate the initial ring cleavage of the bicyclic terpene, is both less extensive and more enigmatic. First referred to as 'ketolactonase-an enzyme for cyclic lactonization'-the enzyme now classified as 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) holds a special place in the history of oxygen-dependent biochemistry, being the first biocatalyst confirmed to undertake a biooxygenation reaction equivalent to the peracid-catalysed Baeyer-Villiger chemical oxidation first reported in the late 19th century. However, following that auspicious beginning, the biochemistry of EC 1.14.14.108, and its isoenzymic partner 3,6-diketocamphane 1,6-monooxygenase (EC 1.14.14.155) was dogged for many years by the mistaken belief that the enzymes were true flavoproteins that function with a tightly-bound flavin cofactor in the active site. This misconception led to a number of erroneous interpretations of relevant experimental data. It is only in the last decade, initially as the result of pure serendipity, that these enzymes have been confirmed to be members of a relatively recently discovered class of oxygen-dependent enzymes, the flavin-dependent two-component monooxygenases. This has promoted a renaissance of interest in the enzymes, resulting in programmes of research that have significantly expanded current knowledge of both their mode of action and regulation in camphor-grown P. putida ATCC 17453. However, some features of the biochemistry of the isoenzymic diketocamphane monooxygenases remain currently unexplained. It is the episodic history of these enzymes and some of what remains unresolved that are the principal subjects of this review.

Dataset Information

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

Publications

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

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