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An Acetyltransferase Conferring Tolerance to Toxic Aromatic Amine Chemicals

MOLECULAR AND FUNCTIONAL STUDIES*
  • Marta Martins
    Footnotes
    Affiliations
    Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS Equipe d'Accueil Conventionée (EAC) 7059, Laboratoire des Réponses Moléculaires et Cellulaires aux Xénobiotiques,

    Unité de Formation et de Recherche (UFR) des Sciences du Vivant
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  • Fernando Rodrigues-Lima
    Affiliations
    Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS Equipe d'Accueil Conventionée (EAC) 7059, Laboratoire des Réponses Moléculaires et Cellulaires aux Xénobiotiques,

    Unité de Formation et de Recherche (UFR) des Sciences du Vivant
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  • Julien Dairou
    Affiliations
    Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS Equipe d'Accueil Conventionée (EAC) 7059, Laboratoire des Réponses Moléculaires et Cellulaires aux Xénobiotiques,

    Unité de Formation et de Recherche (UFR) des Sciences du Vivant
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  • Aazdine Lamouri
    Affiliations
    Laboratoire Interfaces, Traitements, Organisation et Dynamique des Systémes (ITODYS), CNRS UMR 7086

    UFR de Chimie, Université Paris Diderot-Paris 7, 75013 Paris
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  • Fabienne Malagnac
    Affiliations
    Unité de Formation et de Recherche (UFR) des Sciences du Vivant

    Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris-Sud, 91405 Orsay cedex, France
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  • Philippe Silar
    Affiliations
    Unité de Formation et de Recherche (UFR) des Sciences du Vivant

    Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris-Sud, 91405 Orsay cedex, France
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  • Jean-Marie Dupret
    Correspondence
    To whom correspondence should be addressed: Université Paris Diderot-Paris 7, BFA, CNRS EAC 7059, Laboratoire des Réponses Moléculaires et Cellulaires aux Xénobiotiques, Batiment Buffon, 3ème étage, 4, rue Marie Andrée Lagroua Weill Hallé, 75205 Paris Cedex 13, France. Tel.: 33-1-57-27-83-30;
    Affiliations
    Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS Equipe d'Accueil Conventionée (EAC) 7059, Laboratoire des Réponses Moléculaires et Cellulaires aux Xénobiotiques,

    Unité de Formation et de Recherche (UFR) des Sciences du Vivant
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  • Author Footnotes
    * This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC) and la Caisse d'Assurance Maladie des Professions Libérales de Province and running grants from Université Paris Diderot-Paris 7 and Université Paris 11-Sud.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1, supplemental Figs. 1–3, and supplemental references.
    1 Supported by a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.
Open AccessPublished:May 05, 2009DOI:https://doi.org/10.1074/jbc.M109.015230
      Aromatic amines (AA) are a major class of environmental pollutants that have been shown to have genotoxic and cytotoxic potentials toward most living organisms. Fungi are able to tolerate a diverse range of chemical compounds including certain AA and have long been used as models to understand general biological processes. Deciphering the mechanisms underlying this tolerance may improve our understanding of the adaptation of organisms to stressful environments and pave the way for novel pharmaceutical and/or biotechnological applications. We have identified and characterized two arylamine N-acetyltransferase (NAT) enzymes (PaNAT1 and PaNAT2) from the model fungus Podospora anserina that acetylate a wide range of AA. Targeted gene disruption experiments revealed that PaNAT2 was required for the growth and survival of the fungus in the presence of toxic AA. Functional studies using the knock-out strains and chemically acetylated AA indicated that tolerance of P. anserina to toxic AA was due to the N-acetylation of these chemicals by PaNAT2. Moreover, we provide proof-of-concept remediation experiments where P. anserina, through its PaNAT2 enzyme, is able to detoxify the highly toxic pesticide residue 3,4-dichloroaniline in experimentally contaminated soil samples. Overall, our data show that a single xenobiotic-metabolizing enzyme can mediate tolerance to a major class of pollutants in a eukaryotic species. These findings expand the understanding of the role of xenobiotic-metabolizing enzyme and in particular of NATs in the adaptation of organisms to their chemical environment and provide a basis for new systems for the bioremediation of contaminated soils.
      Aromatic amines (AA)
      The abbreviations used are: AA
      aromatic amine
      NAT
      arylamine N-acetyltransferase
      3,4-DCA
      3,4-dichloroaniline
      2-AF
      2-aminofluorene
      4-BOA
      4-butoxyaniline
      WT
      wild type
      HPLC
      high pressure liquid chromatography
      DMSO
      dimethyl sulfoxide.
      3The abbreviations used are: AA
      aromatic amine
      NAT
      arylamine N-acetyltransferase
      3,4-DCA
      3,4-dichloroaniline
      2-AF
      2-aminofluorene
      4-BOA
      4-butoxyaniline
      WT
      wild type
      HPLC
      high pressure liquid chromatography
      DMSO
      dimethyl sulfoxide.
      represent one of the most important classes of occupational or environmental pollutants. Many AA are toxic to most living organisms due to their genotoxic or cytotoxic properties (
      • Kim D.
      • Guengerich F.P.
      ). AA account for 12% of the 415 chemicals that are either known or strongly suspected to be carcinogenic in humans (
      • U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program
      ). AA are common by-products of chemical manufacturing (pesticides, dyestuffs, rubbers, or pharmaceuticals), coal and gasoline combustion, or pyrolysis reactions (
      • Palmiotto G.
      • Pieraccini G.
      • Moneti G.
      • Dolara P.
      ). Moreover, the presence of AA in groundwater or soil samples subject to industrial, agricultural, or urban pollution is of increasing concern, particularly for persistent toxic AA contaminants, such as pesticide-derived anilines (
      • Gan J.
      • Skipper P.L.
      • Gago-Dominguez M.
      • Arakawa K.
      • Ross R.K.
      • Yu M.C.
      • Tannenbaum S.R.
      ).
      The identification of mechanisms by which living organisms can tolerate harmful chemicals, such as AA, is of prime importance to understand their adaptation to stressful environments. In addition, deciphering the molecular mechanisms underlying this tolerance may lead to novel biotechnological and pharmaceutical applications.
      Fungi are environmentally ubiquitous and are found with great diversity in both terrestrial and aquatic environments. Fungi are known to tolerate a large range of chemicals of natural or anthropogenic origin by developing mechanisms to act on xenobiotic and natural compounds (
      • Tortella G.R.
      • Diez M.C.
      • Duran N.
      ,
      • Stadler M.
      • Keller N.P.
      ). Fungi are therefore good models to identify and to understand tolerance mechanisms to xenobiotics (
      • Teeri T.T.
      ,
      • Thakur J.K.
      • Arthanari H.
      • Yang F.
      • Pan S.J.
      • Fan X.
      • Breger J.
      • Frueh D.P.
      • Gulshan K.
      • Li D.K.
      • Mylonakis E.
      • Struhl K.
      • Moye-Rowley W.S.
      • Cormack B.P.
      • Wagner G.
      • Näär A.M.
      ). Moreover, characterization of the mechanisms by which fungi tolerate certain toxic xenobiotics can potentially lead to the identification of new targets for the treatment of fungal infections in vertebrates (
      • Teeri T.T.
      ,
      • Thakur J.K.
      • Arthanari H.
      • Yang F.
      • Pan S.J.
      • Fan X.
      • Breger J.
      • Frueh D.P.
      • Gulshan K.
      • Li D.K.
      • Mylonakis E.
      • Struhl K.
      • Moye-Rowley W.S.
      • Cormack B.P.
      • Wagner G.
      • Näär A.M.
      ) or plants and to the development of new bioremediation tools for cleaning up contaminated environments (
      • Tortella G.R.
      • Diez M.C.
      • Duran N.
      ,
      • D'Annibale A.
      • Rosetto F.
      • Leonardi V.
      • Federici F.
      • Petruccioli M.
      ).
      Using the common ascomycete Podospora anserina as a model, we provide here the demonstration that a single enzyme can mediate tolerance to toxic AA chemicals in a eukaryotic species. This enzyme was identified and characterized as an arylamine N-acetyltransferase (NAT), a xenobiotic-metabolizing enzyme that acetylates efficiently several toxic AA. Targeted disruption of this NAT gene led to the complete loss of tolerance to AA, thus confirming that this enzyme enables the fungus to detoxify AA that would otherwise prove toxic. These findings will help to understand the enzymatic mechanisms contributing to adaptation of living organisms to their environment. In particular, our data demonstrate that the NAT-dependent detoxification mechanisms may provide a eukaryotic organism with tolerance to toxic AA. Moreover, we provide proof-of-principle experiments, using soils contaminated with the highly toxic pesticide residue 3,4-dichloroaniline, proving that the fungal NAT-dependent detoxification pathway may represent a novel model with reasonable cost and a low environmental impact for the bioremediation of AA-contaminated environments.

      Acknowledgments

      We thank Sylvie François for expert technical assistance and Catherine Redeuilh for chemical synthesis.

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