Structural analysis of the reaction cycle of aminoglycoside 6'-N-acetyltransferase type-Ii, an aminoglycoside-resistance factor

Antibiotic resistance is a serious concern in nosocomial bacterial infections. The extensive use of antibiotics has led to the emergence of multidrug-resistant bacteria such as Enterococci. Strains of E. faecium are frequently encountered that are resistant to a diverse range of antibiotics, including beta-lactams, glycopeptides, as well as aminoglycosides. The resistance profile of E. faecium to many aminoglycosides is attributed to an acetyltransferase. The enzyme, aminoglycoside 6'-N-acetyltransferase type-Ii (AAC(6')-Ii), uses acetyl coenzyme A (AcCoA) to modify all clinically relevant aminoglycosides that contain a 6'-amino sugar. Such modifications render aminoglycosides incapable of binding to the 30S bacterial ribosome, thereby preventing antibiotics from inducing protein mistranslation and thus abolishing their bactericidal properties.;Here, we report structural studies for the two remaining states of AAC(6')-Ii. X-ray crystallographic studies of the apo and ternary complex permitted us to describe conformational changes between the different states, define the active site/binding pocket of the enzyme, and rationalize its broad substrate specificity. Complementary solution methods such as nuclear magnetic resonance (NMR), circular dichroism (CD) and small angle X-ray scattering (SAXS) allowed us to gain a better understanding of the dynamics of conformational changes between the apo and complexed states. Estimating quantitative binding energies between the enzyme and several aminoglycosides enabled us to postulate a complete reaction mechanism and extend this mechanism to other acetyltransferases. Ultimately, this detailed structural analysis of AAC(6')-Ii allows us to propose avenues to rejuvenate aminoglycoside therapy against multidrug-resistant pathogens such as E. faecium.;We aimed to explore different possibilities to restore aminoglycoside susceptibility by either protecting aminoglycosides from being modified by AAC(6')-Ii or inhibiting the enzyme so that it can no longer modify aminoglycosides. To this end, we pursued the structural description of the entire AAC(6')-Ii reaction cycle. AAC(6')-Ii employs an ordered sequential mechanism, where AcCoA binds prior to the substrate, and where the modified substrate leaves before coenzyme A (CoASH). Thus, the enzyme cycles through four distinct states: apo, AcCoA complex, ternary complex, and CoASH complex. Two out of the four states (AcCoA and CoASH complexed forms) had already been structurally characterized. In addition, diverse mutagenesis and kinetic studies have determined many aspects of the kinetic mechanism governing the enzyme. However, the most important states (apo and ternary state) remain uncharacterized and no conclusive AAC(6')-Ii reaction mechanism has been proposed.