- Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)
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Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO2) the recalcitrant fused-ring high molecular weight (≥4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.
- Syed, Khajamohiddin,Doddapaneni, Harshavardhan,Subramanian, Venkataramanan,Lam, Ying Wai,Yadav, Jagjit S.
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- Synthesis of 13C4-labelled oxidized metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene
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Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that are implicated in causing lung cancer. BaP is a component of tobacco smoke that is transformed enzymatically to active forms that interact with DNA. We reported previously development of a sensitive stable isotope dilution LC/MS method for analysis of BaP metabolites. We now report efficient syntheses of 13C4-BaP and the complete set of its 13C4-labelled oxidized metabolites needed as internal standards They include the metabolites not involved in carcinogenesis (Group A) and the metabolites implicated in initiation of cancer (Group B). The synthetic approach is novel, entailing use of Pd-catalyzed Suzuki, Sonogashira, and Hartwig cross-coupling reactions combined with PtCl2-catalyzed cyclization of acetylenic compounds. This synthetic method requires fewer steps, employs milder conditions, and product isolation is simpler than conventional methods of PAH synthesis. The syntheses of 13C4-BaP and 13C4-BaP-8-ol each require only four steps, and the 13C-atoms are all introduced in a single step. 13C4-BaP-8-ol serves as the synthetic precursor of all the oxidized metabolites of 13C-BaP implicated in initiation of cancer. The isotopic purities of the synthetic 13C 4-BaP metabolites were estimated to be ≥99.9%.
- Wu, Anhui,Xu, Daiwang,Lu, Ding,Penning, Trevor M.,Blair, Ian A.,Harvey, Ronald G.
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body text
p. 7217 - 7233
(2012/09/05)
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- Synthesis of phenol and quinone metabolites of benzo[a]pyrene, a carcinogenic component of tobacco smoke implicated in lung cancer
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Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. PAHs are present in automobile exhaust and tobacco smoke, and they have recently been designated as human carcinogens. Current ev
- Xu, Daiwang,Penning, Trevor M.,Blair, Ian A.,Harvey, Ronald G.
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experimental part
p. 597 - 604
(2009/06/20)
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- Modulation of cytochrome P4501-mediated bioactivation of benzo[a]pyrene by volatile allyl sulfides in human hepatoma cells.
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Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 microM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 microM DADS and 10-100 microM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 microM of DADS and 10-100 microM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.
- Chun,Kim,Choi
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p. 2205 - 2212
(2007/10/03)
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- Synthesis of the Phenolic Derivatives of Highly Tumorigenic trans-7,8-Dihydroxy-7,8-dihydrobenzopyrene
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Two isomeric phenolic derivatives of trans-7,8-dihydro-7,8-dihydroxybenzopyrene (42), 3,7,8-trihydroxy-trans-7,8-dihydrobenzopyrene (40), and 1,7,8-trihydroxy-trans-7,8-dihydrobenzopyrene (41), have been prepared in order to probe their relevance
- Kumar, Subodh,Kole, Panna L.,Sehgal, Raj K.
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p. 5272 - 5277
(2007/10/02)
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- Synthesis of Potassium Benzo(a)pyren-3-yl Sulfate
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Benzo(a)pyren-3-yl hydrogen sulfate was synthesized from chlorosulphonic acid and benzo(a)pyren-3-ol, and crystallized as the potassium salt.It was hydrolyzed by aryl sulfatase.The UV, IR, and fluorescence spectra of the potassium salt are presented.Keywords - potassium benzo(a)pyren-yl-sulfate; IR spectrum; UV spectrum; enzymatic hydrolysis
- Mori, Masa-aki,Kinoshita, Nadao,Yoshimura, Hidetoshi
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p. 657 - 659
(2007/10/02)
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