Structural Analysis of the Tobramycin and Gentamicin Clinical Resistome Reveals Limitations for Next-generation Aminoglycoside Design

Widespread use and misuse of antibiotics has allowed for the selection of resistant bacteria capable of avoiding the effects of antibiotics. The primary mechanism for resistance to aminoglycosides, a broad-spectrum class of antibiotics, is through covalent enzymatic modification of the drug, waning their bactericidal effect. Tobramycin and gentamicin are two medically important aminoglycosides targeted by several different resistance factors, including aminoglycoside 2 ''-nucleotidyltransferase [ANT(2 '')], the primary cause of aminoglycoside resistance in North America. We describe here two crystal structures of ANT(2 ''), each in complex with AMPCPP, Mn2+, and either tobramycin or gentamicin. Together these structures outline ANT(2 '')'s specificity for clinically used substrates. Importantly, these structures complete our structural knowledge for the set of enzymes that most frequently confer clinically observed resistance to tobramycin and gentamicin. Comparison of tobramycin and gentamicin binding to enzymes in this resistome, as well as to the intended target, the bacterial ribosome, reveals surprising diversity in observed drug target interactions. Analysis of the diverse binding modes informs that there are limited opportunities for developing aminoglycoside analogs capable of evading resistance.