title: 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.
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA.
authors: Ohgew
authors: O
authors: Kweon
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA.
authors: Seong-Jae
authors: SJ
authors: Kim
authors: Department of Biotechnology (BK21 Program) and Institute of Microbiomics, Chung-Ang University, Anseong, Republic of Korea.
authors: Dae-Wi
authors: DW
authors: Kim
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA.
authors: Jeong Myeong
authors: JM
authors: Kim
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA.
authors: Hyun-lee
authors: HL
authors: Kim
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA.
authors: Youngbeom
authors: Y
authors: Ahn
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA.
authors: John B
authors: JB
authors: Sutherland
authors: Division of Microbiology, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA carl.cerniglia@fda.hhs.gov.
authors: Carl E
authors: CE
authors: Cerniglia
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009161:Mycobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011084:Polycyclic Aromatic Hydrocarbons; D010105:Oxygenases