title: Structural and biochemical characterization of an active arylamine N-acetyltransferase possessing a non-canonical Cys-His-Glu catalytic triad.
abstract: 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.
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS EAC4413, 75013 Paris, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Kubiak
authors.forename: Inès
authors.initials: I
authors.lastname: Li de la Sierra-Gallay
authors.forename: Alain F
authors.initials: AF
authors.lastname: Chaffotte
authors.forename: Benjamin
authors.initials: B
authors.lastname: Pluvinage
authors.forename: Patrick
authors.initials: P
authors.lastname: Weber
authors.forename: Ahmed
authors.initials: A
authors.lastname: Haouz
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Dupret
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodrigues-Lima
mesh_terms: D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers; D018698:Glutamic Acid / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D001191:Arylamine N-Acetyltransferase; D003545:Cysteine
keywords: Acetyl Coenzyme A; Crystal Structure; Enzyme Mechanisms; Enzyme Mutation; Enzyme Structure