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- Title
Structural and mechanistic insights into the inhibition of class C β-lactamases through the adenylylation of the nucleophilic serine.
- Authors
Min-Kyu Kim; Young Jun An; Jung-Hyun Na; Jae-Hee Seol; Ju Yeon Ryu; Jin-Won Lee; Lin-Woo Kang; Kyung Min Chung; Jung-Hyun Lee; Jeong Hee Moon; Jong Seok Lee; Sun-Shin Cha; Kim, Min-Kyu; An, Young Jun; Na, Jung-Hyun; Seol, Jae-Hee; Ryu, Ju Yeon; Lee, Jin-Won; Kang, Lin-Woo; Chung, Kyung Min
- Abstract
<bold>Objectives: </bold>: Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C β-lactamases.<bold>Methods: </bold>: The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates.<bold>Results: </bold>: The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1 s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria.<bold>Conclusions: </bold>: Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C β-lactamase inhibitors on the basis of the nucleotide scaffold.
- Subjects
BETA lactamases; ENZYME inhibitors; ADENOSINE monophosphate; SERINE; HYDROLYSIS; BACTERIAL protein metabolism; ANTIBIOTICS; CHEMISTRY; DYNAMICS; HYDROLASES; MICROBIAL sensitivity tests; CEFTAZIDIME; PHARMACODYNAMICS
- Publication
Journal of Antimicrobial Chemotherapy (JAC), 2017, Vol 72, Issue 3, p735
- ISSN
0305-7453
- Publication type
journal article
- DOI
10.1093/jac/dkw491