Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad
| Autor: | Benjamin Pluvinage, Patrick Weber, Inès Li de la Sierra-Gallay, Jean-Marie Dupret, Fernando Rodrigues-Lima, Ahmed Haouz, Xavier Kubiak, Alain Chaffotte |
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| Přispěvatelé: | Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), This work was supported in part by the Université Paris Diderot-Paris 7, Délégation Générale de l'Armement (DGA), the Institut Pasteur Paris, the CNRS and French Infrastructure for Integrated Structural Biology (FRISBI) Grant ANR-10-INSB-05-01. Supported by a fellowship from the Université Paris Diderot-Paris 7. Supported by a fellowship from the DGA., We thank Dr. Karine Moncoq and Ximing Xu for helpful discussions on crystallogenesis assays. We acknowledge the core platform 'Biophysique des macromolécules et de leurs intéractions' (Institut Pasteur) for provision of CD facilities. We also acknowledge Synchrotron Swiss Light Source (Villigen, Switzerland) for the provision of synchrotron radiation facilities, and we thank Meitian Wang for assistance with the use of the Beamline X06DA., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Ribierre, Hélène, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID |
| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular Enzyme Mutation Arylamine N-Acetyltransferase Protein Conformation MESH: Sequence Homology Amino Acid [SDV]Life Sciences [q-bio] Enzyme Mechanisms MESH: Catalytic Domain MESH: Amino Acid Sequence MESH: Base Sequence Crystallography X-Ray Polymerase Chain Reaction Biochemistry MESH: Recombinant Proteins MESH: Histidine MESH: Protein Conformation Catalytic Domain Transferase chemistry.chemical_classification biology Arylamine N-acetyltransferase MESH: Glutamic Acid Recombinant Proteins Enzyme structure [SDV] Life Sciences [q-bio] MESH: Bacillus cereus Protein Structure and Folding Crystal Structure MESH: Models Molecular MESH: DNA Primers endocrine system Stereochemistry Molecular Sequence Data Glutamic Acid Bacillus cereus Acetyl Coenzyme A Catalytic triad Histidine Amino Acid Sequence Cysteine Molecular Biology DNA Primers MESH: Molecular Sequence Data Base Sequence Sequence Homology Amino Acid fungi Active site MESH: Polymerase Chain Reaction Acetyltransferases Cell Biology MESH: Cysteine MESH: Crystallography X-Ray MESH: Arylamine N-Acetyltransferase body regions Enzyme chemistry Acetylation Enzyme Structure biology.protein |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, 2013, 288 (31), pp.22493-22505. ⟨10.1074/jbc.m113.468595⟩ |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.m113.468595 |
| Popis: | International audience; Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms.Background: Catalytic activity of prokaryotic and eukaryotic arylamine N-acetyltransferases (NATs) relies on a strictly conserved catalytic Cys-His-Asp triad.Results: Structural and biochemical studies identified a functional NAT with a Cys-His-Glu catalytic triad.Conclusion: The catalytic triad of these acetyltransferases is more plastic than previously believed.Significance: (BACCR)NAT3 represents the first known functional acetyltransferase with a non-canonical Cys-His-Glu catalytic triad. |
| Databáze: | OpenAIRE |
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