Cosubstrate Tolerance of the Aminoglycoside Resistance Enzyme Eis from Mycobacterium tuberculosis

Abstract

We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6= position is the first to be npropionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal Agents

“…There are three types of AMEs: the aminoglycoside N-acetyltransferases (AACs), the aminoglycoside O-phosphotransferases (APHs), and the aminoglycoside O-nucleotidyltransferases (ANTs). Recently, a unique AAC, the enhanced intracellular survival (Eis) protein from a variety of bacterial species, was found capable of multiacetylating aminoglycosides (12,(28)(29)(30)(31)(32)(33)(34)(35)(36). Knowing that our compounds had good antibacterial activity, we tested the novel C 4 compound to see if it became modified by AMEs (Fig.…”

Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria

“…As we have previously shown, Eis_ Mtb had a very limited cosubstrate tolerance, transferring acyl groups from AcCoA, ProCoA, crotonyl-CoA, and malonyl-CoA only. 12 In addition to the four acyl-CoAs used by Eis_ Mtb , n -butyryl-CoA, n -hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA, and i -valeryl-CoA were tested as potential cosubstrates. All enzymes could only transfer acyl groups from AcCoA and ProCoA, and Eis_ Krh could utilize malonyl-CoA to a lesser extent.…”

“…These results were equal to or greater than the two cases where n -propionylation and acetylation were equal with Eis_ Mtb (KAN and NEO). 12 …”

“… a × indicates that the AG is not a substrate for the enzyme tested. b Data from ref 11. c Data from ref 37. d Data from ref 10. e Data from ref 12. …”

“…37 AACs are known to be cosubstrate promiscuous, 8 although we have shown that Eis_ Mtb has a much more limited cosubstrate tolerance. 12 In addition, we have studied the metabolically possible competition between acetyl-CoA (AcCoA) and n -propionyl-CoA (ProCoA) and the effects it has on multiacetylation of AGs.…”