Redesign of Substrate Specificity and Identification of the Aminoglycoside Binding Residues of Eis from Mycobacterium tuberculosis

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis. Eis from M. tuberculosis (Eis_<i>Mtb</i>) and M. smegmatis (Eis_<i>Msm</i>) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_<i>Mtb</i> is divided into two narrow channels, whereas Eis_<i>Msm</i> contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_<i>Mtb</i>, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_<i>Msm</i>, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_<i>Mtb</i> and for the functional differences from Eis_<i>Msm</i>, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_<i>Mtb</i> by mutating them into their Eis_<i>Msm</i> counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_<i>Mtb</i>. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_<i>Mtb</i> nomenclature) dictate the differences between the acetylation efficiencies of Eis_<i>Mtb</i> and Eis_<i>Msm</i> for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_<i>Mtb</i> into Ala plays a role in APR acetylation