Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance.
Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapen...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
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Elsevier
2021
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| _version_ | 1797069085943529472 |
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| author | Tooke, CL Hinchliffe, P Bonomo, RA Schofield, CJ Mulholland, AJ Spencer, J |
| author_facet | Tooke, CL Hinchliffe, P Bonomo, RA Schofield, CJ Mulholland, AJ Spencer, J |
| author_sort | Tooke, CL |
| collection | OXFORD |
| description | Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (k<sub>cat</sub>), rather than altered K<sub>M</sub> values. High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate. |
| first_indexed | 2024-03-06T22:19:21Z |
| format | Journal article |
| id | oxford-uuid:547ec73a-331f-4431-aba9-12cc915ca086 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T22:19:21Z |
| publishDate | 2021 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | oxford-uuid:547ec73a-331f-4431-aba9-12cc915ca0862022-03-26T16:38:12ZNatural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:547ec73a-331f-4431-aba9-12cc915ca086EnglishSymplectic ElementsElsevier2021Tooke, CLHinchliffe, PBonomo, RASchofield, CJMulholland, AJSpencer, JClass A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (k<sub>cat</sub>), rather than altered K<sub>M</sub> values. High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate. |
| spellingShingle | Tooke, CL Hinchliffe, P Bonomo, RA Schofield, CJ Mulholland, AJ Spencer, J Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. |
| title | Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. |
| title_full | Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. |
| title_fullStr | Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. |
| title_full_unstemmed | Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. |
| title_short | Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. |
| title_sort | natural variants modify klebsiella pneumoniae carbapenemase kpc acyl enzyme conformational dynamics to extend antibiotic resistance |
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