Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

Abstract

The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

Fig. 1.

Multiacetylation of AGs by Eis. Spectrophotometric assay plots monitoring the conversion of various AGs by Eis (black circles) when using one equivalent of AcCoA (A–J), and to their di- (A, E, G, and J), tri- (B, C, D, F, and H), and tetraacetylated (I) counterparts (white circles) when using 10 equivalents of AcCoA.

Fig. 2.

Conversion of NEA to 1,2′,6′-triacetyl-NEA. TLC time course showing the mono-, di-, and triacetylated NEA products generated by Eis using five equivalents of AcCoA. Control reactions for mono- and diacetylation were performed using AAC(2′)-Ic, AAC(3)-IV, and AAC(6′) individually or sequentially.

Fig. 3.

A crystal structure and a proposed mechanism of Eis. (A) A structure of Eis hexamer composed of two trimeric layers as shown in the bottom of this panel. (B) The tripartite fold of an Eis monomer bound to CoA (orange sticks) and acetamide (blue sticks). Residues explored in mutagenesis studies are shown as red sticks. (C) A zoom-in of the Eis active site (as indicated by the rectangle in B). The electrostatic surface of Eis shows the positive charge (blue), negative charge (red), and hydrophobic patches (white). (D) Interactions between Eis, CoA, acetamide, and a proposed catalytic water molecule (magenta). Hydrogen bonds, ionic interactions, and hydrophobic contacts are shown by blue, gray, and green dashed lines, respectively. The carboxylate of Asp260 (magenta) from an adjacent monomer in the hexamer makes a hydrogen bond to the Ppant arm of the CoA and forms salt bridges with Arg93 and Arg94 (in trans) thereby positioning these Arg residues for binding CoA. (E) A proposed mechanism of Eis acetylation.

Fig. 4.

Relative activity of Eis and its mutants toward the ten tested AGs. All activities are normalized against the acetylation of NET by wild-type Eis.