Diketocamphane enantiomer-specific "Baeyer-Villiger" monooxygenases from camphor-grown Pseudomonas putida ATCC 17453

Abstract

Pseudomonas putida ATCC 17453 grew with either (+)-or (-)-camphor as sole carbon source. Enantiomerspecific ' biological Baeyer-Villiger ' monooxygenases were synthesized irrespective of the camphor isomer used for growth. The two enzymes are probably the products of separate genes but showed many similarities. Each consisted of two electrophoretically indentical subunits, bound flavin mononucleotide (FMN) non-covalently and accepted electrons from an induced NADH dehydrogenase which interacted with the FMN bound to the oxygenating component. They showed minor differences in MI with 3,6-diketocamphane 1,6-monooxygenase being the smaller enzyme. Isoelectric focussing showed the two enzymes to have different acidic PI values. Polyclonal antibodies raised against 3,6-diketocamphane 1,6-monooxygenase also cross-reacted with 2,5-diketocamphane 1,2-monooxygenase and its subunits.

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

“…Conversely, the ability of the concentrated (ultrafiltration) major FR contributory activities resulting from the preparative-scale purification (the 27.0 plus 28.5 kDa 'mix', 37.0 and 51.0 kDa native proteins) to facilitate dose-dependent biooxygenation by aliquots of highly purified 2,5-and 3,6-DKCMO oxygen-dependent subunits prepared as described previously (Beecher & Willetts, 1998) was consistently demonstrated using replicate (610) linked enzyme assays (mean data shown in Table 1). Fourthly, there was no evidence that an FR-active NADH oxidase/NADH dehydrogenase characterized as a 36.0 kDa monomer by its sedimentation characteristics and reported by Trudgill and colleagues as the major donor of FMNH 2 to the DKCMO isoenzymes in camphor-grown NCIMB 10007 (Trudgill et al, 1966;Taylor & Trudgill, 1986;Jones et al, 1993) played any significant role in this respect. Even allowing for the possibility that the true molecular mass of the protein reported by Trudgill and colleagues was 43.0 kDa as indicated by its amino acid complement , and as such might correspond to either the 41.0 or 45.0 kDa monomeric activities detected in this study, neither of these possible equivalent FR activities played any significant role in servicing the required FMNH 2 for the DKCMO isoenzymes of camphor-grown NCIMB 10007.…”

“…For many years, studies on the relevant enzyme complement of camphor-grown P. putida NCIMB 10007 (ATCC 17435) have been predicated on activities isolated from early stationary-phase cells (Taylor & Trudgill, 1986;Jones et al, 1993). This despite the fact that prior studies of the first two committed enzymes in the relevant degradative pathway (camphor 5-monooxygenase and the 5-hydroxy camphor dehydrogenase isoenzymes; Fig.…”

“…The first characterized candidate was the monomeric 36 kDa NADH dehydrogenase (Trudgill et al, 1966;Taylor & Trudgill, 1986;Jones et al, 1993), although significantly the purified enzyme had greater NADH : (acceptor) oxidoreductase activity with artificial electron acceptors such as methylene blue . Although the molecular mass of this monomeric protein as assessed by its sedimentation characteristics (Trudgill et al, 1966) was reported to be 36.0 kDa (range 32.0-38.0 kDa), analysis of the amino acid complement of the purified protein suggested that its true molecular mass was higher, at approximately 43 kDa .…”

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

“…However, to date the best-characterised MCMO Type II BVMOs are two enantiocomplementary flavindependent isoenzymes involved in the metabolism of racemic camphor (1) by Pseudomonas putida NCIMB 10007 (= ATCC 17453). The relevant isoenzymes are 2,5-and 3,6-diketocamphane monooxygenase (2, that catalyse the biooxidation of the diketocamphane antipodes derived from (+)-or (-)-camphor to the corresponding lactones, which subsequently undergo spontaneous ring opening thereby triggering further catabolic dissimilation (Conrad et al 1965a;Jones et al 1993;Taylor and Trudgill 1986). The two DKCMOs were recently cloned and recombinantly expressed (Kadow et al 2011; but their exceptional catalytic potential was acknowledged much earlier in a number of studies applying whole cells or partly purified fractions obtained from the NCIMB 10007 strain (Gagnon et al 1994;1995;Grogan et al 1993).…”

“…The two DKCMOs were recently cloned and recombinantly expressed (Kadow et al 2011; but their exceptional catalytic potential was acknowledged much earlier in a number of studies applying whole cells or partly purified fractions obtained from the NCIMB 10007 strain (Gagnon et al 1994;1995;Grogan et al 1993). 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).…”