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The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.
- Source :
-
Antimicrobial agents and chemotherapy [Antimicrob Agents Chemother] 2024 Mar 06; Vol. 68 (3), pp. e0110823. Date of Electronic Publication: 2024 Jan 23. - Publication Year :
- 2024
-
Abstract
- Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both k <subscript>cat</subscript> and K <subscript>M</subscript> such that k <subscript>cat</subscript> / K <subscript>M</subscript> values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both k <subscript>cat</subscript> and K <subscript>M</subscript> is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low K <subscript>M</subscript> value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.<br />Competing Interests: The authors declare no conflict of interest.
- Subjects :
- Cefiderocol
Klebsiella pneumoniae
Hydrolysis
Bacterial Proteins genetics
Bacterial Proteins metabolism
beta-Lactamases genetics
beta-Lactamases metabolism
Drug Combinations
Azabicyclo Compounds pharmacology
Microbial Sensitivity Tests
Ceftazidime pharmacology
Ceftazidime metabolism
Anti-Bacterial Agents pharmacology
Anti-Bacterial Agents metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1098-6596
- Volume :
- 68
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Antimicrobial agents and chemotherapy
- Publication Type :
- Academic Journal
- Accession number :
- 38259088
- Full Text :
- https://doi.org/10.1128/aac.01108-23