Pleiotropic and Epistatic Behavior of a Ring-Hydroxylating Oxygenase System in the Polycyclic Aromatic Hydrocarbon Metabolic Network from Mycobacterium vanbaalenii PYR-1

Abstract

bDespite 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. Mycobacterium vanbaalenii PYR-1 was originally isolated from oil-contaminated estuarine sediment in Redfish Bay, Texas, in 1986 (1-4). It was the first bacterium shown to degrade pyrene, a high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) with four fused benzene rings. This bacterium also degrades other HMW PAHs (fluoranthene, benzo[a]pyrene, benz[a]anthracene, and 7,12-dimethylbenz[a]anthracene) and lowmolecular-weight (LMW) PAHs (naphthalene, fluorene, phenanthrene, and anthracene), primarily via dioxygenation to isomeric cis-dihydrodiols (2, 5-15). Because of its versatile PAH degradation ability, M. vanbaalenii PYR-1 has been extensively studied as a model at both the laboratory and field scales (16, 17). These efforts have produced considerable information on its metabolic, biochemical, physiological, and molecular characteristics, which are also found in other aromatic-compound-degrading bacteria (16)(17)(18)(19)(20)(21)(22).A global PAH metabolic network (MN) in M. vanbaalenii PYR-1 has been proposed on the basis of genomic, proteomic, metabolic, and biochemical information (23). The PAH-MN describes the biochemical pathways for the biodegradation of 10 PAHs with 183 metabolites and 224 chemical rea...

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil

“…Benzo[a]pyrene was not detected in any experimental samples. As discussed below in detail, at the level of PAH metabolism, our proteome analysis revealed that this metabolic disparity from our previous studies in the degradation of the HMW PAH fluoranthene was most likely to have been caused by a differential protein expression pattern, especially of the ring-hydroxylating oxygenase (RHO) systems responsible for the initial hydroxylation of PAH substrates in the ring cleavage process (RCP) functional modules (33,35).…”

“…As a whole, this metabolic capability of M. vanbaalenii PYR-1 is consistent with the results from our prior studies, except for the metabolism of fluoranthene. In our previous biodegradation experiments, M. vanbaalenii PYR-1 always degraded more than 95% of these three PAHs (phenanthrene, pyrene, and fluoranthene) when cultured with single PAH substrates or mixtures of PAHs (33)(34)(35)(43)(44)(45). Benzo[a]pyrene was not detected in any experimental samples.…”

“…The quality and quantity of the proteome results were also evaluated with the previous proteomic data for M. vanbaalenii PYR-1. (For detailed information on proteomics and statistical analysis, see the paper by Kweon et al [35].) In this study, protein abundance was used as a proxy for enzyme activity.…”

“…(i) Sample preparation. The whole-cell proteome of M. vanbaalenii PYR-1 treated with 1% BP crude oil was analyzed as described in our previous studies (33)(34)(35). Briefly, proteins of bacterial cells at each time point were extracted using glass bead homogenization.…”

Effects of Polycyclic Aromatic Hydrocarbon Mixtures on Degradation, Gene Expression, and Metabolite Production in Four Mycobacterium Species

“…Recently, detailed work on initial ring-hydroxylating dioxygenases in Mycobacterium species has led to a more accurate understanding of the diversity of enzymes present, their induction by and transformation rates of PAH substrates, and regioselective product formation (11,42,43,48,60,63,64). nid homologs are subject to induction by different subsets of PAH substrates (11) and seem optimized to be well induced by the PAHs that they can efficiently convert into usable cis-dihydrodiol isomers (i.e., intermediates of mineralization pathways) (42,60).…”