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Mechanistic plasticity in ApmA enables aminoglycoside promiscuity for resistance

Nature Chemical Biology volume 20, pages 234–242 (2024)Cite this article

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Abstract

The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification.

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Fig. 1: Next-generation AG scaffolds vulnerable to 2′-N-acetylation by proteins of two structurally distinct superfamilies.
Fig. 2: ApmA exhibits negative cooperativity of AG acetylation.
Fig. 3: Detail of AG binding in the acetyl-acceptor pocket of ApmA.
Fig. 4: AG resistance conferred by wild-type ApmA and site mutants.
Fig. 5: Proposed role for active site His135 in the acetylation of O3′-containing AGs.

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Data availability

The data that support the findings of this study is available within the main text and its Supplementary Information files; the crystal structures are available under accession codes PDB 7UUJ, 7UUK, 7UUL, 7UUM, 7UUN and 7UUO, with 7JM0 a previously published structure used as a model for molecular replacement. Source data are provided with this paper. Data is also available from the corresponding author upon request.

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Acknowledgements

This work used the Centre for Microbial Chemical Biology core facility at McMaster University for antibiotic susceptibility testing. We thank J. Osipiuk and K. Michalska at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory, for x-ray diffraction data collection. Crystal structures solved in this work were funded in whole or in part with US federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. 75N93022C00035-Center for Structural Biology of Infectious Diseases (CSBID). This research was funded by a Canadian Institutes of Health Research grant (no. FRN-148463), the Ontario Research Fund, and a Canada Research Chair to G.D.W.

Author information

Authors and Affiliations

  1. David Braley Centre for Antibiotics Discovery, M.G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    Emily Bordeleau, Kalinka Koteva & Gerard D. Wright

  2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Peter J. Stogios, Elena Evdokimova & Alexei Savchenko

  3. Center for Structural Genomics of Infectious Diseases (CSGID) University of Calgary, Calgary, Alberta, Canada

    Alexei Savchenko

  4. Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada

    Alexei Savchenko

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  1. Emily Bordeleau
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Contributions

E.B. and G.D.W. conceived the study and designed the experiments. E.B. completed all antimicrobial susceptibility testing and wrote the manuscript. Purification of ApmA and acetylated aminoglycosides was completed by E.B. K.K. completed spectroscopic characterization of acetylated aminoglycosides. P.J.S. solved all crystal structures and wrote the manuscript. E.B. completed structural analysis, protein engineering and steady-state kinetics. E.E. performed protein purification and crystallization. A.S. and G.D.W. oversaw the work and wrote the manuscript.

Corresponding author

Correspondence to Gerard D. Wright.

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The authors declare no competing interests.

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Nature Chemical Biology thanks Mickael Blaise, Hyun Ho Park, Marcelo Tolmasky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Chemical structures of AGs referenced in our study and used for antimicrobial susceptibility testing.

AGs are grouped according to structural subclass with carbons colored to distinguish substitution pattern of 2-DOS ring when present. (a) atypical subclass. (b) 4,6-disubstituted AGs. Kanamycin A and B differ at the 2’ position highlighted in red. Amikacin and plazomicin differ at the N6’ position (red highlight), but both contain the same substituent at N1. (c) 4,5-disubstituted AGs.

Extended Data Fig. 2 Electron density of ligands in six crystal structures of ApmA in this study.

Density shown for aminoglycosides in all panels are simulated annealing omit maps, contoured at 2.5 σ. ApmA wildtype in complex with gentamicin (a), tobramycin (b), kanamycin B (c), paromomycin (d), neomycin (e), ApmA(His135Ala) in complex with tobramycin (f). Simulated annealing omit maps are also shown for CoA contoured at 1.5 σ (in grey). Panel (f) also shows a Polder map for CoA contoured at 3.0 σ (in orange/red) and 2Fo-Fc density for the key active site water molecule contoured at 0.8 σ (cyan).

Extended Data Fig. 3 Additional ligand-bound crystal structure complexes.

(a) Amino acids implicated in positioning kanamycin B. All of the observed AG ligands, (b) tobramycin and (c) kanamycin B, bound to ApmA are superimposed. Each ring is identified with a roman numeral and site of acetylation is labelled. (d) Amino acids implicated in positioning gentamicin C1a. (e) Each gentamicin bound to ApmA is superimposed for analysis of sugar ring conformations within each binding pocket. Panel e also highlights torsional angle differences for glycosidic linkages ΦII/III and ψII/III.

Extended Data Fig. 4 Chemical line representation of Asp144Asn substitution impact on AG binding to ApmA.

Rotamer conformations for functional group of Asp144Asn side chain.

Extended Data Fig. 5 Insert loop region of AG binding pocket is most tolerable of mutation when apramycin is the substrate.

Specific activities for (a) ApmA-mediated acetylation and (b) ApmA(His135Ala)-mediated acetylation of AGs were determined in vitro with 1 M AG substrate, 100 µM acetyl-CoA. Individual data points are shown for two independent replicates. Legend: APR – apramycin, pink circles. TOB – tobramycin, purple diamonds. KAN A – kanamycin A, red hexagons. KAN B – kanamycin B, blue triangles. PAR – paromomycin, yellow squares. Individual data points are shown for two independent replicates. (c) Chemical line drawing representation of apramycin acetylation mediated by His135Ala mutant. (d) Chemical line drawing illustration of tobramycin acetylation mediated by His135Ala mutant.

Source data

Extended Data Table 1 HR-ESI-MS analysis of ApmA-catalyzed acetylated aminoglycosides in positive ion mode
Full size table
Extended Data Table 2 Kinetic parameters for ApmA wild-type and mutants
Full size table
Extended Data Table 3 Aminoglycoside susceptibility testing of E. coli BW25113 ∆tolC∆bamB expressing apmA and apmA mutants under the control of the Pbla promoter
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Tables 1–5.

Reporting Summary

Source data

Source Data Figs. 2 and 5 and Extended Data Fig. 5.

Source kinetics data for Fig. 2b (tab 1) and 2c (tab 2); Fig. 5a and b (tab 3), Fig. 5c and d (tab 4), Extended Data Fig. 5a (tab 5) and Extended Data Fig. 5b (tab 6).

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Bordeleau, E., Stogios, P.J., Evdokimova, E. et al. Mechanistic plasticity in ApmA enables aminoglycoside promiscuity for resistance. Nat Chem Biol 20, 234–242 (2024). https://doi.org/10.1038/s41589-023-01483-3

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