Conferring the Metabolic Self-Sufficiency of the CAM Plasmid of Pseudomonas putida ATCC 17453: The Key Role of Putidaredoxin Reductase

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 Zn(2+), 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.