Deciphering Substrate Promiscuity by Aminoglycoside Resistance Enzymes via a Biophysical Characterization and Dynamics of the Aminoglycoside Acetyltransferase-(3)-IIIb and the Aminoglycoside Phosphotransferase-(3′)-IIIa

Aminoglycoside antibiotics are losing their bactericidal efficacy due to the spread of enzymes that catalyze a covalent modification to them. A common property of many of these aminoglycoside modifying enzymes (AGMEs) is the capacity to modify multiple diverse aminoglycosides thus conferring resistance to these drugs among several pathogenic bacterial species. To gain a better understanding of the protein-antibiotic interactions responsible for resistance and the promiscuous nature of AGMEs, a variety of biophysical techniques including nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), steady state kinetics, intrinsic tryptophan fluorescence, and computational modeling are employed in this work. Results and discussion presented herein are divided into two parts.

In Part I, a detailed thermodynamic and kinetic characterization of the association between the aminoglycoside acetyltransferase-(3)-IIIb (AAC) and several antibiotics and/or coenzyme(s) provides insight into the global properties of the protein. AAC is shown to have a broad substrate range where antibiotic interaction occurs with a favorable enthalpy and unfavorable entropy. When coenzyme A (the non-catalytic form of the acetyl donor, acetyl coenzyme A) is present, enthalpy becomes more favored, entropy more disfavored, and antibiotic affinity significantly increases. AAC shows preference for antibiotics with amine groups at the 2′ and 6′ positions and to those possessing four or more pseudo-saccharide rings. These and other data lay the foundation for understanding AAC and lead into the next discussion wherein the source of promiscuity of AGMEs is explored in Part II.

The aminoglycoside phosphotransferase-(3′)-IIIa (APH), a representative from the phosphotransferase family of AGMEs, has been well characterized previously. However, none of the data presented to date provides rationale for its promiscuity. In this work, NMR derived hydrogen-deuterium exchange experiments reveal that APH maneuvers its entire structure to accommodate diverse antibiotics. Furthermore, presence of an antibiotic creates a more stable APH conformation while coenzyme induces an antibiotic dependent increase in the flexibility of APH. For comparison, a computationally derived homology model of AAC predicts that its promiscuous nature may be due to a large flexible loop. Taken together, APH and AAC, two structurally and functionally diverse proteins, utilize different aspects of structural flexibility to facilitate a broad substrate repertoire that is key to bacterial survival.