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

Abstract

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.

FIG 1

Selection of an M. vanbaalenii PYR-1 Tn5 mutant with an insertion in the pdoA2 gene. (a) Mutant candidates on 7H9 agar plate sprayed with fluorene. The arrows indicate the 6G11 mutant, which was from the 96-well plate, number 6, line G, and row 11, producing no clear zone with fluorene. (b) Diagram showing the Tn5 chromosomal insertion in the pdoA2 gene. KAN, kanamycin resistance.

FIG 2

Growth of wild-type M. vanbaalenii PYR-1 (solid line) and the pdoA2 6G11 mutant (dotted line). SMM broth contains 1% sorbitol supplemented with 200 μM fluorene, anthracene, phenanthrene, pyrene, or fluoranthene. The kinetic values were calculated from the average values of all growth conditions, each of which had triplicate independent cultures.

FIG 3

(a) Single-PAH degradation by cultures of M. vanbaalenii PYR-1 (filled symbols) and the 6G11 mutant (open symbols). (b) Comparisons are shown for individual PAHs only if they were different (0.01< P < 0.07). Cells were grown in SMM supplemented with 1% sorbitol containing 200 μM fluorene, anthracene, phenanthrene, pyrene, or fluoranthene. conc., concentration.

FIG 4

Pleiotropic function of the Pdo system with respect to diverse PAH substrates. Chemical structures of the tested PAH substrates and the corresponding metabolite(s), together with their conversion rates, are presented. The regiospecific information for fluorene and fluoranthene was based on binding modes from the docking simulation. The thickness of the arrow indicates differences in transformation efficiency.

FIG 5

Spatially conserved aromatic amino acids in the substrate-binding pockets of three type V RHO systems, the Pdo system and two Nid systems, in M. vanbaalenii PYR-1 (a) and surface plot of the substrate-binding pocket of the Pdo system with the PAH substrates bound with the highest binding affinity (b). The mononuclear Fe²⁺ and spatially conserved aromatic amino acids are represented as a red ball and a stick model, respectively.

FIG 6

Relative functional activity (RFA) of the RHO members in fluorene, anthracene, phenanthrene, fluoranthene, and pyrene degradation on the basis of their ETC compatibility, substrate specificity, and protein abundance. RHO enzymes were classified according to Kweon's RHO scheme (32). The ETC compatibility of an RHO enzyme was calculated from its RHO classification. Information on protein abundance for RHO enzymes was retrieved from the proteome database of M. vanbaalenii PYR-1 (23). The substrate preference of each RHO enzyme was determined on the basis of the percent conversion rate of each PAH substrate by each enzyme. Please refer to Table S1 in the supplemental material for numerical scores.

FIG 7

Schematic representation of relative contributions of the RHO enzymes to the pathways of fluorene, anthracene, phenanthrene, pyrene, and fluoranthene degradation in M. vanbaalenii PYR-1 (a) and the 6G11 mutant (b). The arrows indicate degradation pathways, and differences in transformation efficiency are represented by arrow thickness. Colored circles indicate RHO enzymes, with the sizes being proportional to the degrees of functional contribution. RCP, ring-cleavage process; SCP, side chain process; CAP, central aromatic process.

FIG 7

Schematic representation of relative contributions of the RHO enzymes to the pathways of fluorene, anthracene, phenanthrene, pyrene, and fluoranthene degradation in M. vanbaalenii PYR-1 (a) and the 6G11 mutant (b). The arrows indicate degradation pathways, and differences in transformation efficiency are represented by arrow thickness. Colored circles indicate RHO enzymes, with the sizes being proportional to the degrees of functional contribution. RCP, ring-cleavage process; SCP, side chain process; CAP, central aromatic process.