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

Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

“…The lower activity of this substrate compared to the substituted bornanone camphor is in agreement with equivalent data obtained by Iwaki et al (2013).…”

“…Although it was repeatedly reported (Iwaki et al 2013) that both DKCMO isoenzymes would exhibit absolute specifity for the respective camphor enantiomer that is the precursor for their physiological substrate, very small amount of product formation from the opposite camphor enantiomer were observed in both cases (Figure 1), data consistent with earlier studies untertaken with these purified enzymes (Beecher 1997;Grogan 1995). …”

“…The molecular weight of this protein as assessed by its sedimentation characteristics has been reported consistently to be 36 kDa, although analysis of the amino acid complement of the purified protein indicated that the true MW was actually 43 kDa (Gunsalus et al 1965); a figure consistent with extensive studies undertaken at the University of Exeter (Beecher 1997;Grogan 1995;McGhie 1998). Genetic exchange studies with the highly transmissible CAM plasmid (Rheinwald et al 1973) led to the assumption that the gene coding for this FR, like those coding for both the DKCMOs, was located on the CAM plasmid, although extensive characterisation of this plasmid has failed repeatedly to identify a relevant locus (Iwaki et al 2013;Koga et al 1989;Palchaudhuri and Chakrabarty 1976). It has been apparent for some time that the validity of a strict functional in vivo relationship between this particular FR and the biooxygenating subunits of the two DKCMO isoenzymes is open to question.…”

“…Both isoenzymes operate in situ as loosely associated trimeric assemblages composed of the biooxygenating component plus a single flavin reductase (FR) (Jones et al 1993). Genes encoding for both the enantio-complementary biooxygenating components (2 x 2,5-DKCMO, 1 x 3,6-DKCMO) are located on the linear 533 kb CAM plasmid (Iwaki et al 2013) and are believed to have evolved from a single progenitor gene by the accepted mechanism of initial gene duplication and subsequent divergence (Horowitz 1945). The crystal structure of the monooxygenase subunit of 3,6-DKCMO has been published (McGhie et al 1998).…”

“…Recently, two significant additional developments challenged further the long-standing proposed functional relationship of the trimeric DKCMOs first proposed by Gunsalus (Conrad et al 1965a;Gunsalus et al 1965) Initially, it was demonstrated that the genes encoding the biooxygenating subunits of both 2,5-and 3,6-DKCMO were functional when expressed in E. coli (Kadow et al 2011;. Subsequently, it has been shown that the biooxygenating components of each DKCMO isoenzyme perform efficient lactone-forming biooxygenation reactions in association with a homodimeric FR (2 x 18 kDa) encoded by the chromosomal DNA of NCIMB 10007 when tandem plasmid constructs carrying both relevant genes were expressed in E. coli (Iwaki et al 2013). …”