title: Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia revealed by crystallographic and small-angle X-ray scattering analysis.
abstract: Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6')-Ie-APH(2'')-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6')-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2'')-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2'')-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6')-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6')-Ie enzyme is joined to APH(2'')-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2'')-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6')-Ie-APH(2'')-Ia enzyme.
authors.affiliation: Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, CA 94025, USA.
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Smith
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Marta
authors.initials: M
authors.lastname: Toth
authors.affiliation: Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, CA 94025, USA.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Weiss
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D000617:Aminoglycosides / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D007612:Kanamycin / Q000737:chemistry; D008958:Models, Molecular; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D000617:Aminoglycosides; D007612:Kanamycin; C108453:6'-aminoglycoside acetyltransferase-2''-aminoglycoside phosphotransferase; D000123:Acetyltransferases; D017853:Phosphotransferases (Alcohol Group Acceptor)
keywords: AAC(6′)-Ie-APH(2′′)-Ia; aminoglycoside-resistance enzyme; small-angle scattering