title: Small-angle X-ray scattering analysis of the bifunctional antibiotic resistance enzyme aminoglycoside (6') acetyltransferase-ie/aminoglycoside (2'') phosphotransferase-ia reveals a rigid solution structure.
abstract: Aminoglycoside (6') acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6')-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5'-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (R(G)) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage.
authors.affiliation: Department of Biochemistry, Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec, Canada.
authors.forename: Shane J
authors.initials: SJ
authors.lastname: Caldwell
authors.forename: Albert M
authors.initials: AM
authors.lastname: Berghuis
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003627:Data Interpretation, Statistical; D003956:Dialysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D008958:Models, Molecular; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D012542:Scattering, Radiation; D013379:Substrate Specificity; D014965:X-Rays
chemical_list: D000123:Acetyltransferases; C056532:aminoglycoside N(6')-acetyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase