Regulation of Camphor Metabolism: Induction and Repression of Relevant Monooxygenases in Pseudomonas putida NCIMB 10007

Abstract

For the first time, the differential rates of synthesis of all the key monooxygenases involved in the catabolism by Pseudomonas putida NCIMB 10007 of bicyclic (rac)-camphor to ∆2,5-3,4,4-trimethylpimelyl-CoA, the first aliphatic pathway intermediate, have been determined to help establish the relevant induction profile of each of the oxygen-dependent enzymes. The efficacy of both relevant substrates and pathway metabolites as inducers has been established. Further, inhibitors with characterised functionality have been used to indicate that the pertinent regulatory controls operate at the level of transcription of the corresponding genes.

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer–Villiger oxidations

“…3,4,[24][25][26] In contrast, two-component systems referred to as type II BVMOs (from group C of FPMOs) are rare. Only two representatives are known so far, 2,5-diketocamphane 1,2-monooxygenase I (2,5-DKCMO; EC 1.14.14.108) and 3,6-diketocamphane 1,6-monooyxgenase (2,6-DKCMO; EC 1.14.14.155), [27][28][29][30][31][32] probably because a twocomponent system is not a trivial biocatalytic arrangement to identify. The mechanism of hydride transfer and the balance of the reductase and oxygenase reactions are still unknown.…”

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

“…That both the growth substrate-dependent and growth phase-dependent changes in the titres of the DKCMOs and the flavin reductases Fred and PdR are illustrative of the wider importance of transcriptional control in up-regulating the pathway for the degradation of alicyclic camphor to aliphatic ∆ 2,5 -3,4,4-trimethylpimelyl-CoA has been confirmed by recent research using the relevant inhibitors rifampicin and actinomycin D [59]. This comprehensive study monitored the differential rates of synthesis of both genome-coded Fred and a number of CAM plasmid-coded activities including the ketolactonases in response to both camphor and key degradation pathway intermediates to establish the various induction and repression circuits that regulate relevant activities in P. putida ATCC 17453 (Figure 8).…”

“…These aspects of absolute stereoselectivity of the DKCMOs contrasts firstly with the known lack of stereoselectivity of both camphor 5- exo hydroxylase [28] and 5- exo hydroxycamphor dehydrogenase [85], the initiating and immediately preceding enzymes respectively in the camphor degradation pathway, and secondly with the fact that both the unstable lactones formed by the enantio-complementary DKCMOs then spontaneously rearrange to the same achiral metabolite OTE (Figure 12). Significantly, any implied potential of P. putida ATCC 17453 to discriminate between (+)- and (−)-camphor as a growth substrate by selectively up-regulating the corresponding stereoselective ketolactonase (Figure 12) is negated by a combination of the established cross-inducibility of the relevant CAM plasmid genes by each of the enantiocomplementary camphor and diketocamphane isomers, and the multivalent product induction of the genes by the shared degradation pathway intermediate OTE ( vide supra , [59]). The very similar nucleotide sequences of the camE 25-1 , camE 25-2 , and camE 36 genes on the CAM plasmid ( vide supra ) suggests that they probably arose by gene duplication and subsequent divergence [86], although any evolutionary advantage of the isoenzymic 2,5-DKCMOs relative to the implicit additional genetic load is difficult to imagine given both the identical outcome reported for highly purified preparations of both recombinantly expressed isoenzymes with the natural substrate (+)-camphor [4], and the reported rarity of the bicyclic monoterpene as a natural substrate in the biosphere [6,33].…”

“…The very similar nucleotide sequences of the camE 25-1 , camE 25-2 , and camE 36 genes on the CAM plasmid ( vide supra ) suggests that they probably arose by gene duplication and subsequent divergence [86], although any evolutionary advantage of the isoenzymic 2,5-DKCMOs relative to the implicit additional genetic load is difficult to imagine given both the identical outcome reported for highly purified preparations of both recombinantly expressed isoenzymes with the natural substrate (+)-camphor [4], and the reported rarity of the bicyclic monoterpene as a natural substrate in the biosphere [6,33]. It may be significant that the camE 25-1 and camE 25-2 genes are located on opposite strands of the CAM plasmid [4], and consequently may possibly be divergently transcribed, although this was not evident from a recent study of transcriptional control of the CAM plasmid-coded enzymes [59]. Interestingly, a comprehensive review has concluded that enantiocomplementary and duplicate enzymes are surprisingly common in Nature, and may in some cases arise serendipitously [87].…”

“…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 ( camE 36 ): E = 2-oxo-∆ 3 -4,5,5-trimethylcyclopentenylacetyl-CoA synthetase ( camF 1 + F 2): F = 2-oxo-∆ 3 -4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase ( camG ) [59]. …”