Abstract
The relative importance of camphor (CAM) plasmid-coded putidaredoxin reductase (PdR) and the chromosome-coded flavin reductases Frp1, Frp2 and Fred for supplying reduced FMN (FNR) to the enantiocomplementary 2,5- and 3,6-diketocamphane monooxygenases (DKCMOs) that are essential for the growth of Pseudomonas putida ATCC 17453 on (rac)-camphor was examined. By undertaking studies in the time window prior to the induction of Fred, and selectively inhibiting Frp1 and 2 with Zn2+, it was confirmed that PdR could serve as the sole active supplier of FNR to the DKCMOs. This establishes for the first time that the CAM plasmid can function as an autonomous extrachromosomal genetic element able to express all the enzymes and redox factors necessary to ensure entry of the C10 bicyclic terpene into the central pathways of metabolism via isobutyryl-CoA.
Highlights
It is well established that bacterial genes specifying catabolic functions against various natural and xenobiotic organic compounds are located on transmissible plasmids (Table 1, [1,2,3,4,5,6,7,8,9,10,11,12]), enabling these extrachromosomal genetic elements to promote both expanded metabolic capabilities of individual species and horizontal genetic transfer of the encoded metabolic competences. These concepts originated from pioneering biochemical and genetic studies conducted in the late 1960s [13] on the large (533 kbp) transmissible camphor (CAM) plasmid, which was shown to be essential for the degradation of (+)-camphor to isobutyryl-CoA in Pseudomonas putida ATCC 17453
While subsequent studies of this bacterium confirmed the location of a number of the genes coding for the requisite enzymes on the large transmissible CAM plasmid [1,15], it is only relatively recently that a more extensive reinvestigation of camphor-grown P. putida ATCC 17453 by Iwaki et al [16] has identified the corresponding plasmid-coded loci for almost all of the enzymes and redox intermediates necessary to metabolise both enantiomers of the C10 bicyclic terpenoid to the level of isobutyryl-CoA (Figure 1)
Iwaki et al.’s comprehensive study was notable for assigning this function to Fred, a chromosome-coded 36 kDa homodimeric flavin reductase FR isolated from cells during the late log to early stationary phase of growth on (+)-camphor [16]
Summary
It is well established that bacterial genes specifying catabolic functions against various natural and xenobiotic organic compounds are located on transmissible plasmids (Table 1, [1,2,3,4,5,6,7,8,9,10,11,12]), enabling these extrachromosomal genetic elements to promote both expanded metabolic capabilities of individual species and horizontal genetic transfer of the encoded metabolic competences. Iwaki et al.’s comprehensive study was notable for assigning this function to Fred, a chromosome-coded 36 kDa homodimeric flavin reductase FR isolated from cells during the late log to early stationary phase of growth on (+)-camphor [16] This was a striking outcome because it served to preclude the ability of the CAM plasmid to function as a catabolically autonomous entity able to promote entry of camphor into the central pathways of metabolism via isobutyryl-CoA. The significance of this newly established role of PdR is that it redresses a significant deficiency of Iwaki et al.’s previous study [1th6a],taitnrdedforresthseesfiarsstigtinmifieciatnctodmepfilceiteensctyheofmIwetaabkoi leitcaplo.’tsepnrtieavlioofutshsetuCdAyM[1p6]la, samndidfotor tfhuencfitriostntaims aenit acuotmonpolemteosutsheexmtreatcahbroolimc poosotemnatilagl oenf tehtiecCeAleMmepnlat sambliedttoo efunnsuctrieonenatsraynoafutthoenCom10obuisceyxctlricactherropmenoesoinmtoal thgeenecetincterlaelmpeantthawblaeytso oenf smureetaenbtorlyisomf thveiaC1is0obbiucytycrliycl-teCropAen. eIinntoortdheercetnotrcaolnpfairthmwathyissofstmateutas,boitlisims imviapeisroabtiuvteytroyld-CemoAo.nIsntroartdeetrhatot Pco. npufitrimdatAhiTsCstCat1u7s4, 5it3icsainmgpreorwatiovne tcoamdepmhoonr-sbtraasteedthmaitnPim. paultimdaedAiTuCmC i1n7t4h5e3 acbansegnrcoewofonfucnacmtiopnhionrg-baacsteivditmieisnifmoralthmeecdhiruommionsothmeea-cbosdenedceeonfzfyumncetsioFnpirn1g, aFcrtpiv2itainesdfForretdh,e wchhricohmiosstohme ep-ucropdoesdeeonfztyhme pesreFspern1t,pFarppe2ra. nd Fred, which is the purpose of the present paper
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