Biochemical and structural analysis of an eis family aminoglycoside acetyltransferase from Bacillus anthracis

Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis-Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis-Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis-Ban displays significant differences from Eis-Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis-Ban. Furthermore, most recently identified inhibitors of Eis-Mtb did not inhibit Eis-Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis-Ban crystal structure. The comparison of the crystal structures of Eis-Ban and Eis-Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.