A Random Sequential Mechanism of Aminoglycoside Acetylation by Mycobacterium tuberculosis Eis Protein

Oleg V Tsodikov,Sylvie Garneau-Tsodikova,Keith D Green,Riccardo Manganelli

doi:10.1371/journal.pone.0092370

Abstract

An important cause of bacterial resistance to aminoglycoside antibiotics is the enzymatic acetylation of their amino groups by acetyltransferases, which abolishes their binding to and inhibition of the bacterial ribosome. Enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Mt) is one of such acetyltransferases, whose upregulation was recently established as a cause of resistance to aminoglycosides in clinical cases of drug-resistant tuberculosis. The mechanism of aminoglycoside acetylation by MtEis is not completely understood. A systematic analysis of steady-state kinetics of acetylation of kanamycin A and neomycin B by Eis as a function of concentrations of these aminoglycosides and the acetyl donor, acetyl coenzyme A, reveals that MtEis employs a random-sequential bisubstrate mechanism of acetylation and yields the values of the kinetic parameters of this mechanism. The implications of these mechanistic properties for the design of inhibitors of Eis and other aminoglycoside acetyltransferases are discussed.

Highlights

The emergence and spread of multidrug-resistant bacteria is a worldwide problem that requires deep understanding of the resistance mechanisms to develop novel rational approaches to antibacterial therapy
We recently demonstrated that a homolog of MtEis from Mycobacterium smegmatis obeys the random sequential mechanism of kanamycin A (KAN) acetylation [6]
In order to distinguish among the two ordered sequential mechanisms and a random sequential mechanism of binding of the AG and the acetyl coenzyme A (AcCoA) to MtEis to form a ternary acetylation complex, we performed a series of steady-state acetylation kinetic assays as a function of two independent variables, the concentrations of AG and AcCoA, both in large excess of the enzyme

Summary

Introduction

The emergence and spread of multidrug-resistant bacteria is a worldwide problem that requires deep understanding of the resistance mechanisms to develop novel rational approaches to antibacterial therapy. We recently demonstrated that a homolog of MtEis from Mycobacterium smegmatis obeys the random sequential mechanism of KAN acetylation [6].

Results

Conclusion