c Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based threedimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane -hydroxylase that oxidized n-alkanes with various chain lengths (C 9 to C 12 and C 15 to C 19 ), as well as alkyl side chains (C 3 to C 9 ) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

A drinking water method for perfluoroalkyl acids (PFAAs) is presented that addresses the occurrence monitoring needs of the U.S. Environmental Protection Agency (EPA) for a future unregulated contaminant monitoring regulation (UCMR). This paper describes the challenges associated with developing an analytical method for 14 PFAAs that will be used for drinking water occurrence monitoring. The method employs solid-phase extraction with analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The final method preservation scheme requires that samples be stored in polypropylene bottles and that they be buffered and free chlorine removed with Trizma buffer. Mean recoveries of chlorinated surface water samples fortified with the PFAAs at 40-100 ng/L (except for the perfluorooctane-sulfonamido-acetic acids at 200 ng/L) are 85-112% with < 5% relative standard deviation. Single laboratory minimum reporting limits of 2.9-14 ng/L are demonstrated with this methodology. The final method meets all of the EPA UCMR survey requirements for sample collection and storage, precision, accuracy, and sensitivity and is expected to be proposed for use under a future UCMR.

1,4-Dioxane has been identified as a probable human carcinogen and an emerging contaminant in drinking water. The United States Environmental Protection Agency's (U.S. EPA) National Exposure Research Laboratory (NERL) has developed a method for the analysis of 1,4-dioxane in drinking water at ng/L concentrations. The method consists of an activated carbon solid-phase extraction of 500-mL or 100-mL water samples using dichloromethane as the elution solvent. The extracts are analyzed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. In the NERL laboratory, recovery of 1,4-dioxane ranged from 94-110% in fortified laboratory reagent water and recoveries of 96-102% were demonstrated for fortified drinking water samples. The relative standard deviations for replicate analyses were less than 6% at concentrations exceeding the minimum reporting level.

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