Kweon, O., Kim, S.-J., Freeman, J. P., Song, J., Baek, S., Cerniglia, C. E.
The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis-dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons.
IMPORTANCE Since the discovery of microbial ring-hydroxylating oxygenases (RHOs) in 1970, the sequences, structures, and enzyme biochemistry, including enantiospecific products, of RHOs have been studied and discussed extensively from the perspective of biodegradation, biotransformation, and biocatalysis processes. However, with all that effort to elucidate the enzymatic mechanisms of RHOs, little is known about the biochemistry and enzymology underlying high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) degradation. We used Mycobacterium vanbaalenii PYR-1 Nid enzymes, the first type V RHO members to display an apparent substrate preference for HMW PAHs. Here, we examine the mechanism of the RHO reaction by integrating structural information of the NidAB and NidA3B3 enzymes with substrate and product data. This study gives us an understanding of how the model RHO systems of M. vanbaalenii PYR-1 metabolize HMW PAHs. The information obtained would also be helpful for successful application of RHO enzymes to the production of industrially and medically important chiral chemicals and the development of PAH bioremediation technologies.