Structure of the bifunctional aminoglycoside‐resistance enzyme AAC(6′)‐Ie‐APH(2′′)‐Ia revealed by crystallographic and small‐angle X‐ray scattering analysis

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 Mg 2 GDP 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.