Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB (2025)

Keywords: bacterial resistance, product-bound complex, ribosome inhibitor, catalytic efficiency, crystal structure

Unique Multiacetylation of Aminoglycosides by Eis.

Eis is a member of a conserved chromosomally encoded family of proteins present in many pathogens (SI Appendix, Fig.S2). It is highly divergent from previously characterized AACs. To gain insight into the mechanism of acetylation of AGs by Eis, we first explored its substrate specificity profile by using a wide set of AGs: netilmicin (NET), sisomicin (SIS), neamine (NEA), ribostamycin (RIB), paromomycin (PAR), neomycin B (NEO), KAN, AMK, tobramycin (TOB), hygromycin (HYG), apramycin (APR), spectinomycin (SPT), and streptomycin (STR). Eis efficiently acetylated a broad variety of AGs (all AGs tested, except APR, SPT, and STR; SI Appendix, Fig.S1) as observed by spectrophotometric (Fig.1 and SI Appendix, TableS3) and mass spectrometry (SI Appendix, TableS2) assays. Remarkably, and to our surprise, Eis catalyzed an unprecedented multiacetylation (di-, tri-, and even tetraacetylation) of its AG substrates. Such multiple acetylation has not been documented for any other known AAC. To confirm the uniqueness of multiacetylation by Eis, we tested six other AACs known to perform monoacetylation at the 2′-, 3-, or 6′ positions (8–16). UV-Visible (UV-Vis) and mass spectrometry assays of these six AACs with the ten AGs that are multiacetylated by Eis, showed only monoacetylation. These reactions were performed under individually optimized conditions with ten equivalents of acetyl-coenzyme A (AcCoA). To assess catalytic specificity of Eis, we measured k_cat and K_m values in steady-state kinetic assays monitoring net acetylation of a variety of AGs (SI Appendix, TableS3). Catalytic efficiencies (k_cat/K_m) varied in a 40-fold range (from 267M^-1s^-1 for AMK to 10,042M^-1s^-1 for NET). Most of this variation was due to differences in k_cat (a 24-fold range), whereas K_m only displayed a fourfold variation, suggesting that Eis evolved to bind different AGs with similar affinities.

Regiospecificity of Triacetylation of NEA by Eis.

To examine the regiospecificity and the potential order of multiacetylation by Eis, we investigated the triacetylation of NEA (Fig.1). NEA contains four primary amines located at positions 1, 2′, 3, and 6′. The reaction progress was monitored by thin-layer chromatography (TLC) (Fig.2 and SI Appendix, TableS4). By comparing the retention factor (R_f) values of the mono-, di-, and triacetylated NEA species formed over time by Eis to those of 2′-, 3-, and 6′-acetyl-NEA obtained by using monoacetylating enzymes, as well as to 6′,2′-, 6′,3-, and 3,2′-diacetyl-NEA obtained by the sequential use of pairs of these enzymes, we demonstrated that Eis triacetylates NEA, first at the 2′-, then at the 6′-, and, finally, at the 1 position. At a qualitative level, Fig.2 demonstrates that these acetylations occur at comparable rates.

Because a purified AAC(1) enzyme was not available, and because no triacetylation of NEA was observed by action of AAC(2′)-Ic, AAC(3)-IV, and AAC(6′) in any order, we could not obtain standards of triacetylated NEA. Thus, we scaled-up the enzymatic triacetylation reaction of NEA to unambiguously establish the specific positions on the NEA scaffold acetylated by Eis. The structure was determined from 1-D and 2-D ¹H and ¹³C NMR spectra of the triacetylated NEA purified by flash chromatography. Corresponding spectra of the nonacetylated NEA were used for comparison. This analysis confirmed acetylation of NEA at the 1-, 2′-, and 6′ positions by Eis (SI Appendix, Figs.S8–S12 and TablesS5 and S6).

Crystal Structure of Eis.

Seeking an understanding of the catalytic mechanism of Eis, we determined a crystal structure of Eis in complex with the reaction products CoA and acetylated HYG, at a resolution of 1.95Å (Figs.3 A–C and SI Appendix, Figs.S13, S14, and TableS7). Eis forms a tightly packed hexamer, in agreement with its hexameric state in solution (SI Appendix, Fig.S4). The hexamer resembles a sandwich of two threefold symmetrical trimers (Fig.3A). Overall, 3,800Ų of solvent-accessible surface area of each monomer is buried in the hexameric interface (17), indicating extensive intimate contacts between the monomers. The Eis monomer consists of three regions that are assembled into a heart-shaped molecule (Fig.3B). This shape is formed by an unusual fusion of two general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT) regions and a C-terminal region (SI Appendix, Fig.S13). The GNAT fold is common among N-acetyltransferases in all kingdoms of life. A clearly distinguishable CoA and an acetamide moiety of acetylated HYG in the electron density in the N-terminal GNAT region of Eis are positioned analogously to those found in other AACs (8, 18). This observation indicates that this region is involved in catalysis. Despite our efforts of crystallization of Eis with many different AGs under a variety of conditions, we did not observe defined electron density for the rest of the AG molecule, reflecting its multiple orientations or flexibility when bound in the active site. The central region of Eis, also resembling a GNAT fold, lacks conserved Arg residues that bind CoA phosphates in this superfamily and, therefore, it is unlikely to be catalytically active. The juxtaposition of the N-terminal and the central GNAT regions creates a large and intricate AG binding pocket (Figs.3 B and C). The C-terminal region, which consists of a 5-stranded β-sheet flanked by four helices on one side, resembles the fold of the animal sterol carrier protein (SCP) (19, 20). However, it lacks the hydrophobic cavity used by SCP to bind lipids. This organization enables the C-terminal peptide of Eis (residues 392–402) to wedge between the two GNAT regions and reach into the active site.

Mechanism of Acetylation by Eis.

The bound CoA and the acetamide reveal a network of ionic, hydrophobic, and hydrogen bonding interactions that stabilize the cosubstrate in the Eis active site pocket, and indicate a likely acetylation mechanism (Fig.3D). This mechanism is very similar to that proposed for 2′-N-acetylation by AAC(2′)-Ic (8). The AG amino group to be acetylated is ideally positioned by the His119 backbone for a direct nucleophilic attack on the CoA thioester. The tetrahedral transition state is stabilized by polarization of the thioester carbonyl through hydrogen bonding to Phe84 and Val85. As proposed for the N-myristoylation mechanism by Nmt1p (21), the C-terminal carboxylate of Phe402 interacts with the amino group through a bridging water molecule and likely serves as a remote base. We propose that the universally conserved Tyr126 at a distance of 3.4Å from the sulfhydryl group of CoA (3.6Å in the case of the 2′-N-acetylation) likely serves as a general acid to protonate the CoA thiolate.

To probe this mechanism in solution, we first investigated the importance of the proposed catalytic residues Phe402 and Tyr126 (Fig.4 and SI Appendix, Figs.S2, S5, and TableS2). Either the addition of a CHis₆ tag or the removal of the three C-terminal amino acid residues (Eis1–399) nearly abolished the acetylation activity of Eis on all AGs tested. These observations confirmed the important role of the C-terminal tail and the Phe402 residue in catalysis. A deletion mutant lacking the C-terminal region (Eis1–311) and the C-terminal region alone (Eis292–402) were also inactive, as expected. The presence of the central GNAT region of Eis indicated a formal possibility that Eis contained a second active site in this region, even though AcCoA coordinating residues were not present. The mutant of the proposed catalytic acid Tyr126Ala completely abolished the acetylation activity on all substrates tested, which agreed with its proposed role in the mechanism and ruled out the possibility of the existence of another active site. We next probed the importance of His119 involved in coordination of the proposed catalytic water molecule (Fig.3E). Mutation of this residue to an Ala resulted in a decrease in activity and a change in the number of acetylated sites (mono- vs. di- vs. tri-) of all AGs tested, thereby confirming its key role in Eis action. Complete inactivity of the Tyr310Ala mutant of the hydrophobic core of Eis indicated that structural stability is crucial for catalytic activity of the enzyme. Similarly, mutating either Trp197 in the hydrophobic core or Asp292 partially buried in the interface of the two GNAT folds to an Ala, almost completely eradicated Eis activity. As another control, we generated an Ala mutant of the surface-exposed Arg148 located far away from any important surface or interface. As expected, we found that this mutation had no significant effect on the Eis activity.

A close examination of the AG binding pocket reveals that it is formed by the adjoining surfaces of the two GNAT folds (Fig.3C). This deep and bifurcated AG binding pocket must allow for AGs of different sizes and structures to be accommodated. The surface of the binding pocket and its entrances are highly negatively charged with occasional hydrophobic patches, which must ensure electrostatic attraction to amine-rich substrates. The residues that line the binding pocket are marked in SI Appendix, Fig.S2.

We next inspected the effects of residues lining the AG and AcCoA binding surfaces on the Eis activity by point mutagenesis (Fig.4 and SI Appendix, Fig.S5 and TableS2). Interestingly, although the Phe24Ala mutant drastically decreased acetylation activity, mutations of the neighboring Phe27, Ile28, and Trp36 to Ala did not affect the efficiency of the acetylation reactions, but instead resulted in a change in the number of acetylated sites of many of the AGs tested. These observations are in agreement with the proposed role of these residues in AG binding. In addition, we confirmed that Val87, Arg93, Ser121, and Arg128 all play an important role in AcCoA binding as their mutations to Ala led to a general decrease in catalytic activity of Eis. Finally, the Ala mutation of Phe84 whose backbone amide is proposed to stabilize the oxyanion of the acetyl group of AcCoA and whose side chain is buried in the hydrophobic core, resulted in both a large catalytic deficiency and a change in the number of acetylated sites of most AGs studied.

The biological function of Eis in Mtb has been the subject of recent interest (6, 7, 22–29). The biochemical and structural studies described in this report provide clear evidence for an unprecedented multiacetylation capability of Eis that inactivates AG antibiotics. Eis homologues are found in a variety of pathogens that have developed resistance to AGs. Upregulation of eis genes in these bacteria may confer resistance to AGs, as observed in M. tuberculosis, although such studies have not yet been reported. The unique and efficient strategy of deactivation of AGs by multiacetylation presented herein may be a general, widespread resistance mechanism, and yet another evolutionary hurdle to overcome.

Expression and Purification of Eis and Eis Mutants.

The eis gene (gene locus Rv2416c) was PCR amplified from genomic DNA of M. tuberculosis H37Rv and cloned into pET28a. Wild type, Eis mutants, and l-selenomethionine (SeMet)-Eis were overexpressed in BL21(DE3) cells and purified by Ni²⁺ and size-exclusion chromatography. A detailed description of the cloning, mutants generation, Eis expression, and purification can be found in the SI Appendix, Figs.S3–S4 and TableS1.

Spectrophotometric Measurements of Acetyltransferase Activity.

The acetyltransferase activity of Eis proteins was monitored by a UV-Vis assay in which the free thiol group of CoA, generated by enzyme catalyzed reaction, is coupled to 4,4′-dithiodipyridine to produce thiopyridone monitored by increase in absorbance at 324nm (ε₃₂₄=19,800M^-1cm^-1), as described in detail in SI Appendix, Fig.S5. The measurements of k_cat and K_m values for net acetylation of AGs by Eis were performed by this method, as described in detail in SI Appendix, TableS3.

Determination of Number of Sites Acetylated on each AG by Eis Proteins.

The number of acetylations was estimated by the above UV-Vis assay and then determined accurately by liquid chromatography mass spectrometry (SI Appendix, TableS2). Representative mass spectra are shown in SI Appendix, Fig.S6.

Structure Determination of Triacetylated NEA by TLC and NMR.

The specific sites for triacetylation of NEA by Eis were directly visualized by comparison with single- and double-acetylated NEA standards (SI Appendix, TableS4) produced by regiospecific AACs: AAC(2′)-Ic from Mtb (8, 9) (SI Appendix, Fig.S7), AAC(3)-IV from Escherichia coli (4, 5), AAC(3)-Ib and AAC(6′)-Ib′ from the bifunctional AAC(3)-Ib/AAC(6′)-Ib′ from Pseudomonas aeruginosa (12, 13), AAC(6′) from the bifunctional AAC(6′)/AG phosphotransferase (APH)(2′′)-Ia from Staphylococcus aureus (10, 14), and AAC(6′)-IId from the bifunctional AG nucleotidyltransferase (ANT)(3′′)-Ii/AAC(6′)-IId from Serratia marcescens (15, 16). A detailed description of the TLC conditions can be found in the SI Appendix.

The exact acetyl positions on triacetyl-NEA were confirmed by ¹H, ¹³C, 2-D-total correlation spectroscopy (TOCSY), 2-D-COSY, distortionless enhancement by polarization transfer (DEPT), and heteronuclear correlation (HETCOR) NMR. Proton connectivities were assigned using 2-D-TOCSY and 2-D-COSY spectra. Signals of all carbons were derived from HETCOR and DEPT spectra. The details are given in SI Appendix, Figs.S8–S12 and Tables S5–S6.

Crystallization and Structure Determination of the Eis-CoA-Acetamide Complex.

Crystals of SeMet-Eis-CoA (grown in the presence of KAN) and Eis-CoA-acetylated HYG (grown with KAN, but subsequently exchanged to HYG in the presence of AcCoA) were grown by the hanging drop method and subsequently flash frozen in liquid nitrogen. Five different AGs were tried in the growth and soaking of the crystals and yielded similar electron density in the Eis active site. The best-diffracting crystals were obtained by using KAN/CoA during growth and HYG/AcCoA during soaking. The crystal structure was determined by the Se single anomalous dispersion method and refined at 1.95Å resolution for the Eis-CoA-acetylated HYG complex. The details of crystallization and structure determination are described in the SI Appendix, Figs.S13–S14 and TableS7.

Supporting Information

• 1.Caminero JA, Sotgiu G, Zumla A, Migliori GB. Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis. Lancet Infect Dis. 2010;10:621–629. doi: 10.1016/S1473-3099(10)70139-0. [DOI] [PubMed] [Google Scholar]
• 2.Houghton JL, Green KD, Chen W, Garneau-Tsodikova S. The future of aminoglycosides: The end or renaissance? ChemBioChem. 2010;11:880–902. doi: 10.1002/cbic.200900779. [DOI] [PubMed] [Google Scholar]
• 3.Carter AP, et al. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature. 2000;407:340–348. doi: 10.1038/35030019. [DOI] [PubMed] [Google Scholar]
• 4.Moazed D, Noller HF. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature. 1987;327:389–394. doi: 10.1038/327389a0. [DOI] [PubMed] [Google Scholar]
• 5.Steitz TA. A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol. 2008;9:242–253. doi: 10.1038/nrm2352. [DOI] [PubMed] [Google Scholar]
• 6.Campbell PJ, et al. Molecular detection of mutations associated with first and second-line drug resistance compared with conventional drug susceptibility testing in M. tuberculosis. Antimicrob Agents Chemother. 2011;55:2032–2041. doi: 10.1128/AAC.01550-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Zaunbrecher MA, Sikes RD, Jr, Metchock B, Shinnick TM, Posey JE. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 2009;106:20004–20009. doi: 10.1073/pnas.0907925106. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Vetting MW, Hegde SS, Javid-Majd F, Blanchard JS, Roderick SL. Aminoglycoside 2′-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates. Nat Struct Biol. 2002;9:653–658. doi: 10.1038/nsb830. [DOI] [PubMed] [Google Scholar]
• 9.Hegde SS, Javid-Majd F, Blanchard JS. Overexpression and mechanistic analysis of chromosomally encoded aminoglycoside 2′-N-acetyltransferase (AAC(2′)-Ic) from Mycobacterium tuberculosis. J Biol Chem. 2001;276:45876–45881. doi: 10.1074/jbc.M108810200. [DOI] [PubMed] [Google Scholar]
• 10.Green KD, Chen W, Houghton JL, Fridman M, Garneau-Tsodikova S. Exploring the substrate promiscuity of drug-modifying enzymes for the chemoenzymatic generation of N-acylated aminoglycosides. ChemBioChem. 2010;11:119–126. doi: 10.1002/cbic.200900584. [DOI] [PubMed] [Google Scholar]
• 11.Magalhaes ML, Blanchard JS. The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli. Biochemistry. 2005;44:16275–16283. doi: 10.1021/bi051777d. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Dubois V, et al. Molecular characterization of a novel class 1 integron containing bla(GES-1) and a fused product of aac3-Ib/aac6′-Ib′ gene cassettes in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2002;46:638–645. doi: 10.1128/AAC.46.3.638-645.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Kim C, Villegas-Estrada A, Hesek D, Mobashery S. Mechanistic characterization of the bifunctional aminoglycoside-modifying enzyme AAC(3)-Ib/AAC(6′)-Ib′ from Pseudomonas aeruginosa. Biochemistry. 2007;46:5270–5282. doi: 10.1021/bi700111z. [DOI] [PubMed] [Google Scholar]
• 14.Boehr DD, Daigle DM, Wright GD. Domain-domain interactions in the aminoglycoside antibiotic resistance enzyme AAC(6′)-APH(2′′) Biochemistry. 2004;43:9846–9855. doi: 10.1021/bi049135y. [DOI] [PubMed] [Google Scholar]
• 15.Centron D, Roy PH. Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion. Antimicrob Agents Chemother. 2002;46:1402–1409. doi: 10.1128/AAC.46.5.1402-1409.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Kim C, Hesek D, Zajicek J, Vakulenko SB, Mobashery S. Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3′′)-Ii/AAC(6′)-IId from Serratia marcescens. Biochemistry. 2006;45:8368–8377. doi: 10.1021/bi060723g. [DOI] [PubMed] [Google Scholar]
• 17.Tsodikov OV, Record MT, Jr, Sergeev YV. Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J Comput Chem. 2002;23:600–609. doi: 10.1002/jcc.10061. [DOI] [PubMed] [Google Scholar]
• 18.Maurice F, et al. Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance. EMBO Rep. 2008;9:344–349. doi: 10.1038/embor.2008.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Choinowski T, Hauser H, Piontek K. Structure of sterol carrier protein 2 at 1.8Å resolution reveals a hydrophobic tunnel suitable for lipid binding. Biochemistry. 2000;39:1897–1902. doi: 10.1021/bi992742e. [DOI] [PubMed] [Google Scholar]
• 20.Dyer DH, et al. The structural determination of an insect sterol carrier protein-2 with a ligand-bound C16 fatty acid at 1.35-A resolution. J Biol Chem. 2003;278:39085–39091. doi: 10.1074/jbc.M306214200. [DOI] [PubMed] [Google Scholar]
• 21.Bhatnagar RS, Futterer K, Waksman G, Gordon JI. The structure of myristoyl-CoA:protein N-myristoyltransferase. Biochim Biophys Acta. 1999;1441:162–172. doi: 10.1016/s1388-1981(99)00155-9. [DOI] [PubMed] [Google Scholar]
• 22.Wei J, et al. Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol. 2000;182:377–384. doi: 10.1128/jb.182.2.377-384.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Dahl JL, Wei J, Moulder JW, Laal S, Friedman RL. Subcellular localization of the Iitracellular survival-enhancing Eis protein of Mycobacterium tuberculosis. Infect Immun. 2001;69:4295–4302. doi: 10.1128/IAI.69.7.4295-4302.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Roberts EA, Clark A, McBeth S, Friedman RL. Molecular characterization of the eis promoter of Mycobacterium tuberculosis. J Bacteriol. 2004;186:5410–5417. doi: 10.1128/JB.186.16.5410-5417.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Dahl JL, et al. The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression. J Bacteriol. 2005;187:2439–2447. doi: 10.1128/JB.187.7.2439-2447.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Samuel LP, et al. Expression, production, and release of the Eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion. Microbiology. 2007;153:529–540. doi: 10.1099/mic.0.2006/002642-0. [DOI] [PubMed] [Google Scholar]
• 27.Lella RK, Sharma C. Eis (enhanced intracellular survival) protein of Mycobacterium tuberculosis disturbs the cross regulation of T-cells. J Biol Chem. 2007;282:18671–18675. doi: 10.1074/jbc.C600280200. [DOI] [PubMed] [Google Scholar]
• 28.Wu S, et al. Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth. Microbiology. 2009;155:1272–1281. doi: 10.1099/mic.0.024638-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 29.Shin DM, et al. Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. PLoS Pathog. 2010;6:e1001230. doi: 10.1371/journal.ppat.1001230. [DOI] [PMC free article] [PubMed] [Google Scholar]

Supplementary Materials