PHENOL HYDROXYLASE CLONED FROM RALSTONIA-EUTROPHA STRAIN E2 EXHIBITS NOVEL KINETIC-PROPERTIES

Ralstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol- degrading bacterium expressing phenol-oxygenating activity with a low K-s (the apparent half-saturation constant in Haldane's equation) and an extremely high K-SI (the apparent inhibition constant). To identify the molecular basis for these novel cellular kinetic properties, a 9. 5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl(-) Ca t(+)) to grow on phenol as the sole carbon source was cloned from stra in E2 into plasmid pRO1614. PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase. The cloned genes consist of eight complete ORFs, designated poxRABCDEFG. The products are homologo us to those of dmpRKLIMNOPQ of Pseudomonas sp. CF600 sharing 30-65% id entity: this suggests that the phenol hydroxylase is a multicomponent enzyme, the kinetic constants for phenol-oxygenating activity of PAO1c (pROE217) were determined, and these were compared with those of strai n E2. The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined. A comparison of these kineti c data suggests that phenol hydroxylase, the first enzyme in the pheno l-degradative pathway, determines K-s and K-SI values for the cellular phenol-oxygenating activity. It is thus suggested that the phenol hyd roxylase cloned from strain E2 exhibits the novel kinetic properties t hat were observed with intact cells of strain E2.