Characterised Flavin-Dependent Two-Component Monooxygenases from the CAM Plasmid of Pseudomonas putida ATCC 17453 (NCIMB 10007): ketolactonases by Another Name

Abstract

The CAM plasmid-coded isoenzymic diketocamphane monooxygenases induced in Pseudomonas putida ATCC 17453 (NCIMB 10007) by growth of the bacterium on the bicyclic monoterpene (rac)-camphor are notable both for their interesting history, and their strategic importance in chemoenzymatic syntheses. Originally named 'ketolactonase-an enzyme system for cyclic lactonization' because of its characterised mode of action, (+)-camphor-induced 2,5-diketocamphane 1,2-monooxygenase was the first example of a Baeyer-Villiger monooxygenase activity to be confirmed in vitro. Both this enzyme and the enantiocomplementary (-)-camphor-induced 3,6-diketocamphane 1,6-monooxygenase were mistakenly classified and studied as coenzyme-containing flavoproteins for nearly 40 years before being correctly recognised and reinvestigated as FMN-dependent two-component monooxygenases. As has subsequently become evident, both the nature and number of flavin reductases able to supply the requisite reduced flavin co-substrate for the monooxygenases changes progressively throughout the different phases of camphor-dependent growth. Highly purified preparations of the enantiocomplementary monooxygenases have been exploited successfully for undertaking both nucleophilic and electrophilic biooxidations generating various enantiopure lactones and sulfoxides of value as chiral synthons and auxiliaries, respectively. In this review the chequered history, current functional understanding, and scope and value as biocatalysts of the diketocamphane monooxygenases are discussed.

Keywords: diketocamphane monooxygenase; enantiocomplementary enzymes; enantiodivergent biotransformation; flavin-dependent two-component monooxygenase; ketolactonase.

Figure 1

Pathway of (+)- and (−)-camphor degradation in P. putida ATCC 17453. A = cytochrome P450 monooxygenase (camCAB): B = exo-hydroxycamphor dehydrogenase (camD): C = 2,5-diketocamphane 1,2-monooxygenase (camE_25-1 + camE_25-2): D = 3,6-diketocamphane 1,6-monooxygenase (camE₃₆): E = 2-oxo-∆³-4,5,5-trimethylcyclopentenylacetyl-CoA synthetase (camF₁ + F₂); F = 2-oxo-∆³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (camG): FNR = reduced flavin mononucleotide: Fred = 36 kDa chromosome-coded flavin reductase: PdR = putidaredoxin reductase subunit of cytochrome P45O monooxygenase (camA): Frp 1 + 2 = chromosome-coded ferric reductases: diatomic oxygen molecules participating in the four monooxygenase-catalysed steps is shown in green, as in each case are the fates of each component oxygen atom.

Figure 2

Localization of additional genes and predicted open reading frames (ORFs) flanking the established initial genes of the camphor camDCAB operon and its repressor, camR, on an ~40.5-kb sequenced region of the CAM plasmid of P. putida ATCC 17453. The predicted ORFs or genes are numbered from 1 to 27, except for the established camRDCAB genes, which are shaded in black. The orientation of the arrows indicates the direction of gene transcription. The candidate genes of this study (camE_25-1, camE_25-2, and camE₃₆) representing the three diketocamphane monooxygenase (DKCMO) isozymes are highlighted in gray. The previously established OTEMO-encoding gene has been designated camG in accordance with the respective catabolic steps. camS, -T, -U, and -V are potential transcriptional regulators; camV is a close homolog of camR. The black solid line represents the previously sequenced region, with the indicated GenBank accession numbers. Figure 2 reproduced from Iwaki et al. [4], with the permission from American Society for Microbiology: licence number 4487631509592.

Figure 3

(A) Proposed [25] functional complex between E₁ (NADH oxidase) and E₂ (2,5-diketocamphane 1,2-monooxygenase); (B) Proposed [25] reaction sequence for the combined action of E₁ and E₂.

Figure 3

(A) Proposed [25] functional complex between E₁ (NADH oxidase) and E₂ (2,5-diketocamphane 1,2-monooxygenase); (B) Proposed [25] reaction sequence for the combined action of E₁ and E₂.

Figure 4

Speculative ‘metabolic grid’ proposed for the early metabolism of (+)-camphor [24,25,27] based on the characterised lactonizing reactions catalysed by partially purified E₂ (2,5-diketocamphane 1,2-monooxygnase).

Figure 5

Specific activity of total flavin reductase (FR) throughout the growth of P. putida ATCC 17543 on either (+)-camphor, (−)-camphor or succinate as sole carbon source.

Figure 6

Changes in the optical density (A₅₀₀nm), succinate (mM), (rac)-camphor (mM), and the specific activity of key enzymes of (rac)-camphor degradation during diauxic growth of P. putida ATCC 17543 on succinate plus (rac)-camphor-based defined medium.

Figure 7

Relative contribution of the different assayed FNR-generating enzymes to the total flavin reductase activity titre throughout the various phases of (rac)-camphor-dependent growth of P. putida ATCC 17543 [18].

Figure 8

Transcriptional controls of the pathway of (+)- and (−)-camphor degradation in P. putida ATCC 17453 ---●+ = induction: -●--- = repression: A = cytochromeP450 monooxygenase (camCAB): B = exo-hydroxycamphor dehydrogenase (camD): C = 2,5-diketocamphane 1,2-monooxygenase (cam_25-1 + cam_25-2): D = 3,6-diketocamphane 1,6-monooxygenase (camE₃₆): E = 2-oxo-∆³-4,5,5-trimethylcyclopentenylacetyl-CoA synthetase (camF1 + F2): F = 2-oxo-∆³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (camG) [59].

Figure 9

Protein sequence alignment of the oxygenating subunits of 2,5-DKCMO-1 (orf-4, camE_25-1), 2,5-DKCMO-2 (orf-22, camE_25-2) and 3,6-DKCMO (orf-19, camE₃₆). Amino acid residues: common to 2,5-DKCMO-1, 2,5-DKCMO-2, and 3,6-DKCMO = black; common to 2,5-DKCMO-1 and 2,5-DKCMO-2 = purple; exclusive to 2,5-DKCMO-1 = green; exclusive to 2,5-DKCMO-2 = red; exclusive to 3,6-DKCMO = blue.

Figure 10

Rationale for superimposing the minimalised structures of the sulfoxides formed by 2,5-DKCMO and 3,6-DKCMO, and the resultant total dimensions (‘cubic space’) of the relevant enzyme active sites.

Figure 11

Contrasting outcomes of the lactonization of (rac)-bicyclo[3.2.0]hept-2-en-6-one by chemical oxidation (metachlorperbenzoic acid, MCPBA) and biotransformation (2,5-DKCMO); e.e.% = enantiomeric excess.

Figure 12

The relationships between nonselective and stereoselective enzymes that constitute the pathway for the degradation of (rac)-camphor to OTE in P. putida ATCC 17453, and the roles of relevant chiral and achiral molecules as substrates and transcriptional regulators. A = induction by (+)-camphor and (−)-camphor, plus product induction by OTE: B = induction by (+)-camphor and (−)-camphor, plus product induction by 2,5-DKC and 3,6-DKC: C = induction by (+)-camphor, (−)-camphor and 2,5-DKC, plus cross-induction by 3,6-DKC, and product induction by OTE: D = induction by (+)-camphor, (−)-camphor and 3,6-DKC, plus cross-induction by 2,5-DKC, and product induction by OTE: A + B + C + D = ‘from the top’ coordinate induction by (+)-camphor and (−)-camphor.

Figure 13

(A). Generation of enantiocomplementary substituted cyclopentanes with two chiral centres by deploying DKCMO-catalysed biotransformations of unsubstituted (rac)-bornan-2-one as a key step. (B). Generation of enantiocomplementary substituted cyclopentanes with four chiral centres by deploying DKCMO-catalysed biotransformations of (rac)-5′,7′-disubstituted bicyclo[2.2.1] ketones as a key step.

Figure 14

Biotransformation of the tricyclic ketone (rac)-tricyclo[4.2.1.0]nonan-2-one by highly purified preparations of native 2,5-DKCMO and 3,6-DKCMO; e.e. = enantiomeric excess.