Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase
KPC-2 (<i>Klebsiella pneumoniae</i> 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 i...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
American Chemical Society
2023
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| _version_ | 1797110506246373376 |
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| author | Tooke, CL Hinchliffe, P Beer, M Zinovjev, K Colenso, CK Schofield, CJ Mulholland, AJ Spencer, J |
| author_facet | Tooke, CL Hinchliffe, P Beer, M Zinovjev, K Colenso, CK Schofield, CJ Mulholland, AJ Spencer, J |
| author_sort | Tooke, CL |
| collection | OXFORD |
| description | KPC-2 (<i>Klebsiella pneumoniae</i> 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 (<i>k</i><sub>cat</sub>), 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-(2<i>R</i>) 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-(2<i>R</i>) 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-(2<i>R</i>) 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. |
| first_indexed | 2024-03-07T07:55:48Z |
| format | Journal article |
| id | oxford-uuid:48579c89-8869-4839-bb2d-200872542508 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:55:48Z |
| publishDate | 2023 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | oxford-uuid:48579c89-8869-4839-bb2d-2008725425082023-08-22T13:19:02ZTautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamaseJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:48579c89-8869-4839-bb2d-200872542508EnglishSymplectic ElementsAmerican Chemical Society2023Tooke, CLHinchliffe, PBeer, MZinovjev, KColenso, CKSchofield, CJMulholland, AJSpencer, JKPC-2 (<i>Klebsiella pneumoniae</i> 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 (<i>k</i><sub>cat</sub>), 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-(2<i>R</i>) 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-(2<i>R</i>) 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-(2<i>R</i>) 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. |
| spellingShingle | Tooke, CL Hinchliffe, P Beer, M Zinovjev, K Colenso, CK Schofield, CJ Mulholland, AJ Spencer, J Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase |
| title | Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase |
| title_full | Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase |
| title_fullStr | Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase |
| title_full_unstemmed | Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase |
| title_short | Tautomer-specific deacylation and Ω-loop flexibility explain the carbapenem-hydrolyzing broad-spectrum activity of the KPC-2 β-lactamase |
| title_sort | tautomer specific deacylation and ω loop flexibility explain the carbapenem hydrolyzing broad spectrum activity of the kpc 2 β lactamase |
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