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=-[␤,␥-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.

Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

“…23,24 Sulfonamides 39 and 46 did not inhibit KAN acetylation by the AACs tested at concentrations as high as 200 μ M, with the exception of 39 , which inhibited 15% of the AAC(2′)-Ic activity when tested at 200 μ M. These data demonstrate the high selectivity of our inhibitors toward Eis.…”

Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens

“…In early attempts to understand the role and impact of domain interactions of bifunctional AMEs on antibiotic resistance, Wright and co-workers demonstrated that a connecting α-helix between the two domains of the fused AAC(6′)-Ie/APH(2″)-Ia was critical to maintain both correct structure and function of both domains [21]. More recently, by using small-angle X-ray scattering to model the structure of this bifunctional enzyme, Berghuis and co-workers concluded that AAC(6′)-Ie/APH(2″)-Ia adopts a rigid conformation in solution, which likely contributes to improvement of the enzymatic activities of the individual enzymes [22]. By revisiting the nucleotide and AG substrate specificity of this bifunctional enzyme, Vakulenko and co-workers recently reported that, contrary to previous beliefs, GTP is the exclusive phosphate donor in cells for this enzyme [9].…”

Mechanisms of resistance to aminoglycoside antibiotics: overview and perspectives

“…5A). Also, bifunctional enzymes, such as AAC(6′)-Ie/APH(2″)-Ia from Staphylococcus aureus , 109–112 AAC(3)-Ib/AAC(6′)-Ib′ from P. aeruginosa , 113, 114 ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens , 115–117 and AAC(6′)-30/AAC(6′)-Ib from P. aeruginosa 118, 119 exist and are capable of multiple types of AG modification. AAC(6′)-Ib is the most prevalent and clinically relevant AME; approximately fifty variants of AAC(6′)-Ib exist in numerous Gram-negative species.…”