Pleiotropic metabolic behavior of a ring-hydroxylating oxygenase Pdo system in the polycyclic aromatic hydrocarbon-metabolic network from Mycobacterium vanbaalenii PYR-1

We conducted a forward genetics mutagenesis to select mutants of Mycobacterium vanbaalenii PYR-1, which are unable to degrade low-molecular-weight polycyclic aromatic hydrocarbons (LMW PAHs) but still degrade 4-ringed PAHs. A PAH spray method produced a mutant, 6G11, which appeared to lose its ability to degrade fluorene and anthracene. This mutant was identified to be defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO), which has been reported to be responsible for the initial step of phenanthrene oxidation. To understand its pleiotropic behavior in the PAH-metabolic network (MN), we analyzed PAH metabolic and proteomic profiles of strain 6G11 and conducted a PAH biotransformation study for the PdoA2 enzyme using an E, coli expression system. The mutant 6G11 showed a substantial decrease in the rate of degradation of fluorene, anthracene, and pyrene and a differential color change in the culture media containing fluorene, phenanthrene, and fluoranthene. The proteome study revealed a PAH substrate-based regulation of the PdoA2 enzyme. Consistent with these observations, the recombinant PdoA2 oxidized a wide range of substrates with a relatively high conversion rate of naphthalene, fluorene, anthracene, and phenanthrene. Taken together, the results provided direct evidence not only for the in vivo functional role of PdoA2 in LMW PAH hydroxylation but also for the robustness of the PAH-MN to this genetic perturbation. This study expanded our knowledge on the mechanism of PAH degradation, which would help developing better strategies for the removal of carcinogenic PAHs from ecosystems and the reduction of health risks associated with exposure to PAHs.