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
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 kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM 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 KM 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.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2024 |
|---|---|
| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:68 |
|---|---|
| Enthalten in: |
Antimicrobial agents and chemotherapy - 68(2024), 3 vom: 06. März, Seite e0110823 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Birgy, Andre [VerfasserIn] |
|---|
| Links: |
|---|
| Anmerkungen: |
Date Completed 07.03.2024 Date Revised 08.03.2024 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1128/aac.01108-23 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM367495740 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM367495740 | ||
| 003 | DE-627 | ||
| 005 | 20240308232351.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 240123s2024 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1128/aac.01108-23 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1320.xml |
| 035 | |a (DE-627)NLM367495740 | ||
| 035 | |a (NLM)38259088 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Birgy, Andre |e verfasserin |4 aut | |
| 245 | 1 | 4 | |a 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 |
| 264 | 1 | |c 2024 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 07.03.2024 | ||
| 500 | |a Date Revised 08.03.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM 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 KM 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a KPC-31 β-lactamase | |
| 650 | 4 | |a KPC-33 β-lactamase | |
| 650 | 4 | |a antibiotic resistance | |
| 650 | 4 | |a cross-resistance | |
| 650 | 4 | |a enzyme variants | |
| 650 | 4 | |a transient kinetics | |
| 650 | 4 | |a β-lactamase | |
| 650 | 7 | |a Ceftazidime |2 NLM | |
| 650 | 7 | |a 9M416Z9QNR |2 NLM | |
| 650 | 7 | |a carbapenemase |2 NLM | |
| 650 | 7 | |a EC 3.5.2.6 |2 NLM | |
| 650 | 7 | |a Anti-Bacterial Agents |2 NLM | |
| 650 | 7 | |a Cefiderocol |2 NLM | |
| 650 | 7 | |a SZ34OMG6E8 |2 NLM | |
| 650 | 7 | |a Bacterial Proteins |2 NLM | |
| 650 | 7 | |a beta-Lactamases |2 NLM | |
| 650 | 7 | |a EC 3.5.2.6 |2 NLM | |
| 650 | 7 | |a Drug Combinations |2 NLM | |
| 650 | 7 | |a Azabicyclo Compounds |2 NLM | |
| 700 | 1 | |a Nnabuife, Christina |e verfasserin |4 aut | |
| 700 | 1 | |a Palzkill, Timothy |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Antimicrobial agents and chemotherapy |d 1972 |g 68(2024), 3 vom: 06. März, Seite e0110823 |w (DE-627)NLM000026506 |x 1098-6596 |7 nnns |
| 773 | 1 | 8 | |g volume:68 |g year:2024 |g number:3 |g day:06 |g month:03 |g pages:e0110823 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1128/aac.01108-23 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 68 |j 2024 |e 3 |b 06 |c 03 |h e0110823 | ||