Research ArticleHEALTH AND MEDICINE

A low-barrier hydrogen bond mediates antibiotic resistance in a noncanonical catalytic triad

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Science Advances  04 Apr 2018:
Vol. 4, no. 4, eaas8667
DOI: 10.1126/sciadv.aas8667
  • Fig. 1 Binary complexes of AAC-VIa.

    (A) Surface representation of AAC-VIa–sisomicin (green carbon atoms) binary complex superimposed on the enzyme–acetyl-CoA (yellow carbon atoms) binary complex. The AAC-VIa surface cavities that bind sisomicin and coenzyme A (CoASH) are colored black and gray, respectively, whereas the catalytic triad is shown in magenta. (B) Close-up view of sisomicin (green carbon atoms) interaction with AAC-VIa. The site of antibiotic acetylation is circled in red. (C) Close-up view of acetyl-CoA (yellow carbon atoms) interaction with AAC-VIa. The acetyl donor is circled in red. Hydrogen bonds are represented as black dashed lines, and water molecules are shown as red spheres. The active-site residues have carbon atoms colored magenta. (D) Sisomicin with the site of acetylation (the N3 position of the unprimed ring) circled.

  • Fig. 2 Room temperature neutron structure of AAC-VIa.

    Close-up view of the neutron diffraction model and nuclear density for sisomicin-bound AAC-VIa. Sisomicin-bound active-site residues are colored with magenta carbon atoms, and sisomicin with green carbon atoms. His189 from the neutron diffraction data acquired with the apo-enzyme is shown and colored with tan carbon atoms. The 2Fo-Fc nuclear density map is shown in light gray. The Fo-Fc deuterium nuclear omit map for the proton involved in the LBHB is shown in green. Hydrogen bonds are represented as red dashed lines.

  • Fig. 3 Proposed AAC-VIa mechanism.

    (A) Proposed catalytic mechanism of AAC-VIa–mediated acetylation of sisomicin. (B) Close-up view of the active site of the ternary enzyme-CoASH-sisomicin complex superimposed on the binary enzyme–acetyl-CoA complex. Active-site residues are colored with magenta carbon atoms, CoASH is colored with orange carbon atoms, acetyl-CoA is colored with yellow carbon atoms, and sisomicin is colored with green carbon atoms. (C) Close-up view of the active site of the enzyme–acetyl sisomicin complex superimposed on the binary enzyme–acetyl-CoA complex. Active-site residues are colored with magenta carbon atoms, acetyl-CoA is colored with gray carbon atoms, and acetylated sisomicin is colored with green carbon atoms. Hydrogen bonds are represented as red dashed lines.

  • Fig. 4 Product-mediated cofactor release.

    Superposition of the acetylated sisomicin (gray carbon atoms) structure in the binary enzyme–acetyl sisomicin complex on the ternary enzyme-CoASH-sisomicin (cyan protein carbon atoms, with sisomicin carbon atoms colored green and CoASH carbon atoms colored orange) structure. The acetylation of sisomicin leads to flipping of the adjacent sugar ring concomitant with active-site rearrangement that would lead to loss of CoASH product hydrogen bonds.

  • Table 1 Data collection and refinement statistics.

    RMS, root mean square.

    ApoSisomicinCoATernaryAcetyl-CoAAcetylated sisomicin
    Resolution range50.0 to 2.2
    (2.27 to 2.20)
    50.0 to 1.8
    (1.86 to 1.80)
    50.0 to 2.2
    (2.28 to 2.20)
    50.0 to 2.05
    (2.12 to 2.05)
    50.0 to 2.05
    (2.12 to 2.05)
    50.0 to 2.2
    (2.27 to 2.20)
    Space groupC 2P 61P 61P 61P 61C 2
    Unit cell
      a, b, and c (Å)89.0, 86.0, and 51.178.6, 78.6, and 83.678.1, 78.1 and 83.377.5, 77.5, and 83.978.5, 78.5, and 83.984.4, 86.9, and 50.3
      α, β, and γ (°)90, 120.0, and 9090, 90, and 12090, 90, and 12090, 90, and 12090, 90, and 12090, 119.8, and 90
    Unique reflections15,39326,88014,07718,00418,24014,753
    Multiplicity*2.4 (2.2)2.9 (2.8)5.6 (4.9)2.9 (2.7)2.6 (2.1)3.0 (2.6)
    Completeness (%)90.5 (94.7)98.7 (96.3)95.1 (72.2)99.3 (98.2)98.6 (97.0)91.8 (97.3)
    Mean I/σ(I)8.8 (2.4)12.0 (2.4)23.0 (3.8)11.0 (2.0)11.6 (2.3)10.4 (3.2)
    Wilson B-factor37.416.5432.731.5823.0239.2
    R-merge0.084 (0.504)0.089 (0.532)0.091 (0.484)0.10 (0.602)0.101 (0.526)0.094 (0.554)
    Reflections used in
    refinement
    15,38826,85114,07317,98418,23314,750
    Reflections used for R-free7711302688899905737
    R-work0.236 (0.314)0.149 (0.227)0.176 (0.208)0.189 (0.289)0.179 (0.275)0.282 (0.450)
    R-free0.267 (0.333)0.186 (0.286)0.212 (0.289)0.207 (0.278)0.205 (0.288)0.308 (0.496)
    Number of nonhydrogen
    atoms
      Macromolecules202820892042204820432028
      Ligands13548795135
      Solvent5139812918527457
    RMS (bonds)0.0020.0090.0020.0020.0020.004
    RMS (angles)0.50.90.60.50.50.9
    Ramachandran favored (%)95.197.096.096.297.096.6
    Ramachandran allowed (%)4.13.03.63.42.63.4
    Ramachandran outliers (%)0.80.00.40.40.40.0
    Clashscore753226
    Average B-factor441933312351
      Macromolecules441733312251
      Ligands432060323253
      Solvent423036342949
    PDB code6BC66BC76BC56BC36BC46BC2

    *Number in parentheses represents values in the highest-resolution shell.

    • Table 2 Room temperature data collection and refinement statistics.
      ApoSisomicin
      X-rayNeutronX-rayNeutron
      Resolution range50.0 to 2.0 (2.07 to 2.00)15.0 to 2.3 (2.38 to 2.30)50.0-1.9 (1.97 to 1.90)15.0 to 2.20 (2.28 to 2.20)
      Space groupI 2I 2
      Unit cell
        a, b, and c (Å)51.6, 86.1, and 78.850.8, 86.2, and 76.9
        β (°)94.094.5
      Unique reflections22,33614,02925,87415,519
      Multiplicity*3.4 (3.4)2.8 (2.2)1.7 (1.6)2.8 (2.5)
      Completeness (%)97.4 (95.3)91.3 (84.3)99.3 (97.2)92.5 (89.3)
      Mean I/sigma(I)11.5 (3.1)11.6 (3.0)17.2 (3.1)10.4 (3.3)
      R-merge0.069 (0.390)0.154 (0.287)0.047 (0.326)0.147 (0.360)
      Reflections used in refinement22,33614,02925,86715,512
      Reflections used for R-free11267081286763
      R-work (%)15.922.115.019.8
      R-free (%)1924.618.122.0
      Number of non-hydrogen atoms
        Macromolecules20292055
        Ligands031
        Solvent88126
      RMS (bonds)0.0100.013
      RMS (angles)0.9340.81
      Ramachandran favored (%)97.096.6
      Ramachandran allowed (%)3.03.0
      Ramachandran outliers (%)0.00.4
      Clashscore2.51.8
      Average B-factor
        Macromolecules55.752.9
        Ligands53.9
        Solvent58.259.1
      PDB code6BBR6BB2

      *Number in parentheses represents values in the highest-resolution shell.

      Supplementary Materials

      • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaas8667/DC1

        fig. S1. Overall structure of apo acetyltransferase AAC-VIa.

        fig. S2. Kinetics and homologs of AAC-VIa.

        fig. S3. The binary AAC-VIa–CoASH complex.

        fig. S4. Overlay of ternary enzyme-CoASH-sisomicin complex with binary complexes of the enzyme with CoASH, acetyl-CoA, and sisomicin.

        fig. S5. Sisomicin-mediated ordering of acetyl-CoA binding site.

      • Supplementary Materials

        This PDF file includes:

        • fig. S1. Overall structure of apo acetyltransferase AAC-VIa.
        • fig. S2. Kinetics and homologs of AAC-VIa.
        • fig. S3. The binary AAC-VIa–CoASH complex.
        • fig. S4. Overlay of ternary enzyme-CoASH-sisomicin complex with binary complexes of the enzyme with CoASH, acetyl-CoA, and sisomicin.
        • fig. S5. Sisomicin-mediated ordering of acetyl-CoA binding site.

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