Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase

Catherine L Tooke; Philip Hinchliffe; Michael Beer; Kirill Zinovjev; Charlie K Colenso; Christopher J Schofield; Adrian J Mulholland; James Spencer

doi:10.1021/jacs.2c12123

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase

Catherine L Tooke, Philip Hinchliffe, Michael Beer, Kirill Zinovjev, Charlie K Colenso, Christopher J Schofield, Adrian J Mulholland, James Spencer^*

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

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Abstract

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Original language	English
Pages (from-to)	7166-7180
Number of pages	15
Journal	Journal of the American Chemical Society
Volume	145
Issue number	13
Early online date	27 Mar 2023
DOIs	https://doi.org/10.1021/jacs.2c12123
Publication status	Published - 5 Apr 2023

Bibliographical note

Funding Information:
Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [no. BB/J014400/1] studentship to C.L.T. and no. BB/T008741/1 studentship to M.B.). C.L.T, J.S., A.J.M., and C.J.S. thank the Medical Research Council (no. MR/T016035/1). C.J.S. thanks the Medical Research Council and the Wellcome Trust for funding. A.J.M. thanks the U.K. Engineering and Physical Science Research Council (EPSRC grant no. EP/M022609/1) for support. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. X-ray diffraction data were collected at the BL13-XALOC beamline at the ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposal nos. 172122 and 23269) and the staff of beamlines I24 and I04 for assistance. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol (http://www.bristol.ac.uk/acrc/).

Funding Information:
Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [no. BB/J014400/1] studentship to C.L.T. and no. BB/T008741/1 studentship to M.B.). C.L.T, J.S., A.J.M., and C.J.S. thank the Medical Research Council (no. MR/T016035/1). C.J.S. thanks the Medical Research Council and the Wellcome Trust for funding. A.J.M. thanks the U.K. Engineering and Physical Science Research Council (EPSRC grant no. EP/M022609/1) for support. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. X-ray diffraction data were collected at the BL13–XALOC beamline at the ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposal nos. 172122 and 23269) and the staff of beamlines I24 and I04 for assistance. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol ( http://www.bristol.ac.uk/acrc/ ) .

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

Keywords

AMR

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Mechanism, Dynamics and Inhibition of Class A β-Lactamases through Computation and Experiment
Beer, M. (Author), Spencer, J. (Supervisor) & Mulholland, A. J. (Supervisor), 4 Feb 2025
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)

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@article{c1ddb86c11e54b029062d77f3e36a9db,

title = "Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase",

abstract = "KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 {\AA}) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.",

keywords = "AMR",

author = "Tooke, {Catherine L} and Philip Hinchliffe and Michael Beer and Kirill Zinovjev and Colenso, {Charlie K} and Schofield, {Christopher J} and Mulholland, {Adrian J} and James Spencer",

note = "Funding Information: Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [no. BB/J014400/1] studentship to C.L.T. and no. BB/T008741/1 studentship to M.B.). C.L.T, J.S., A.J.M., and C.J.S. thank the Medical Research Council (no. MR/T016035/1). C.J.S. thanks the Medical Research Council and the Wellcome Trust for funding. A.J.M. thanks the U.K. Engineering and Physical Science Research Council (EPSRC grant no. EP/M022609/1) for support. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. X-ray diffraction data were collected at the BL13-XALOC beamline at the ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposal nos. 172122 and 23269) and the staff of beamlines I24 and I04 for assistance. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol (http://www.bristol.ac.uk/acrc/). Funding Information: Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [no. BB/J014400/1] studentship to C.L.T. and no. BB/T008741/1 studentship to M.B.). C.L.T, J.S., A.J.M., and C.J.S. thank the Medical Research Council (no. MR/T016035/1). C.J.S. thanks the Medical Research Council and the Wellcome Trust for funding. A.J.M. thanks the U.K. Engineering and Physical Science Research Council (EPSRC grant no. EP/M022609/1) for support. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. X-ray diffraction data were collected at the BL13–XALOC beamline at the ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposal nos. 172122 and 23269) and the staff of beamlines I24 and I04 for assistance. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol ( http://www.bristol.ac.uk/acrc/ ) . Publisher Copyright: {\textcopyright} 2023 The Authors. Published by American Chemical Society.",

year = "2023",

month = apr,

day = "5",

doi = "10.1021/jacs.2c12123",

language = "English",

volume = "145",

pages = "7166--7180",

journal = "Journal of the American Chemical Society",

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Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase. / Tooke, Catherine L ; Hinchliffe, Philip ; Beer, Michael et al.
In: Journal of the American Chemical Society, Vol. 145, No. 13, 05.04.2023, p. 7166-7180.

Research output: Contribution to journal › Article (Academic Journal) › peer-review

TY - JOUR

T1 - Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase

AU - Tooke, Catherine L

AU - Hinchliffe, Philip

AU - Beer, Michael

AU - Zinovjev, Kirill

AU - Colenso, Charlie K

AU - Schofield, Christopher J

AU - Mulholland, Adrian J

AU - Spencer, James

N1 - Funding Information: Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [no. BB/J014400/1] studentship to C.L.T. and no. BB/T008741/1 studentship to M.B.). C.L.T, J.S., A.J.M., and C.J.S. thank the Medical Research Council (no. MR/T016035/1). C.J.S. thanks the Medical Research Council and the Wellcome Trust for funding. A.J.M. thanks the U.K. Engineering and Physical Science Research Council (EPSRC grant no. EP/M022609/1) for support. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. X-ray diffraction data were collected at the BL13-XALOC beamline at the ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposal nos. 172122 and 23269) and the staff of beamlines I24 and I04 for assistance. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol (http://www.bristol.ac.uk/acrc/). Funding Information: Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [no. BB/J014400/1] studentship to C.L.T. and no. BB/T008741/1 studentship to M.B.). C.L.T, J.S., A.J.M., and C.J.S. thank the Medical Research Council (no. MR/T016035/1). C.J.S. thanks the Medical Research Council and the Wellcome Trust for funding. A.J.M. thanks the U.K. Engineering and Physical Science Research Council (EPSRC grant no. EP/M022609/1) for support. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. X-ray diffraction data were collected at the BL13–XALOC beamline at the ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposal nos. 172122 and 23269) and the staff of beamlines I24 and I04 for assistance. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol ( http://www.bristol.ac.uk/acrc/ ) . Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.

PY - 2023/4/5

Y1 - 2023/4/5

N2 - KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

AB - KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

KW - AMR

U2 - 10.1021/jacs.2c12123

DO - 10.1021/jacs.2c12123

M3 - Article (Academic Journal)

C2 - 36972204

SN - 0002-7863

VL - 145

SP - 7166

EP - 7180

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 13

ER -

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase

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Mechanism, Dynamics and Inhibition of Class A β-Lactamases through Computation and Experiment

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