Direct evidence to support the new status of 2,5-DKCMO then followed from a study undertaken by a multinational research group led by Peter Lau that deployed genomic tools to further characterise the biochemistry of some of the key enzymes of the camphor biodegradation pathway of
P. putida ATCC 17453
[49]. Notably, the group published a seminal 2013 study which specifically achieved two principal goals with respect to 2,5-DKCMO
[50]. Firstly, for the first time, the complete sequence of the DNA of the 533 kb double-stranded linear CAM plasmid
[12][13][14][15] was established, and homology searching was used to assign functions to every one of its
orfs. This was an important outcome because it confirmed directly that the plasmid encodes relevant genes for all the characterised enzymes of both the (+)-camphor (2,5-DKCMO-dependent) and (-)-camphor (3,6-DKCMO-dependent) degradation pathways (
Figure 2). Further, for the first time it became apparent that 2,5-DKCMO functions as a mixture of two very similar isoenzymic forms (60% overall similarity) coded for by two genes (
camE25-1 and
camE25-2) located on opposite stands of the plasmid. Secondly, the 2013 study identified and characterised Fred, a chromosomal DNA-coded 2 × 18 kDa homodimeric nonflavoprotein reductase that reduced FMN as an external substrate (
Figure 5).
6. As the Area of Light Expands, So Does the Perimeter of Darkness
Further insights into the newly recognised status of 2,5-DKCMO as an fd-TCMO then emerged from subsequent studies that scrutinised the nature and extent of those FR activities able to support the ketolactonase throughout the successive trophophasic and idiophasic stages of growth of
P. putida ATCC 17453 in a camphor-based minimal medium
[51][52]. While supporting the finding of the 2013 report
[50] that Fred was an inducible chromosome-coded dimeric (2 × 18 kDa) nonflavoprotein reductase, these new investigations revealed that it was only present in detectible levels in late trophophase (primary metabolism) and subsequent idiophase (secondary metabolism). In this respect, it resembles the equivalent ‘tailoring FRs’ involved in the late stage synthesis of some antibiotics
[53][54], and a wide range of other microbial secondary metabolites
[55][56][57][58][59][60]. Circumstantial evidence also suggests that Fred may correspond to the NADH oxidase activity of camphor-grown
P. putida ATCC 17453 previously reported by both Gunsalus
[27][28][29][30][31][61][62][63][64][65][66] and Trudgill
[67][68][69][70][71][72][73], who both focussed their research exclusively camphor-grown cells entering idiophasic growth, a time frame chosen specifically to optimise biomass yield
[72]. Trudgill conclusively confirmed by two different methods that the MW of the active form of the enzyme was 36 kDa, but significantly never subjected it to SDS-PAGE analysis.
Conversely, the equivalent role throughout trophophase (primary metabolism) was fulfilled by a combination of three newly recognised competent reductases. Two of these, Frp1 (27.0 kDa) and Frp2 (28.5 kDa), were constitutive monomeric nonflavoprotein ferric (flavin) reductases coded for by chromosomal DNA. Structurally, both corresponded closely with other well-characterised ferric (flavin) reductases and the related ferrodoxin reductases, a group of functionally related FRs that have been confirmed to be widely distributed in other prokaryotes
[74][75].
By way of contrast, the third relevant activity after extensive purification was confirmed by sequence homology to be putidaredoxin reductase (PdR, MW 45.6 kDa), an inducible flavoprotein coded for by the
camA gene on the CAM plasmid, and characterised by a tightly bound FAD cofactor
[76][77][78][79]. This particular reductase was already known to function as one of the three activities (
camABC) that collectively serve as cytochrome P450 monooxygenase (cytP450MO), the well-studied multienzyme complex that hydroxylates camphor
[80][81][82], thereby initiating the terpene biodegradation pathway in
P. putida ATCC 17453 (
Figure 2).
The proven ability of PdR to satisfy the FMNH
2 requirement of 2,5-DKCMO has additional strategic significance because it supports the concept that this key bio-oxygenating enzyme, responsible for initiating the ring cleavage of the bicyclic terpene (+)-camphor, can function independently of the chromosomal DNA of
P. putida ATCC 17453. This contrasts with the previous proposals made by both Gunsalus and Trudgill that the monooxygenase is dependent on a 36 kDa chromosome-coded NADH oxidase as its source of FMNH
2 (vide supra). This aspect of the functional independence of 2,5-DKCMO from chromosomal DNA was then supported further by using the known inhibitory effect of Zn
2+ on ferric (flavin) reductases
[83][84][85][86].
Currently there still remain a number of incompletely understood or unresolved aspects of the bio-oxygenating activity of 2,5-diketocamphane 1,2-monooxygenase from camphor-grown P. putida ATCC 17453. One important issue is to develop a more comprehensive understanding of the biochemical logistics that enable PdR to serve both as a redox partner for 2,5-DKCMO and as an integral member of the trimeric cytP450MO bio-oxygenating consortium (Figure 6).
Figure 6. Schematic of the two contrasting roles of putidaredoxin reductase (PR) in camphor-grown P. putida ATCC 17453. Reaction 1 is common to both roles. In both cases, the sequence of reactions progresses from one to three. P450MO = cytochrome P450 mono-oxygenase; Pdx = putidaredoxin; DKCMO = 2,5-diketocamphane 1,2-mono-oxygenase; DKC = 2,5-diketocamphane.