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Structural and mechanistic insights into the inhibition of class C beta-lactamases through the adenylylation of the nucleophilic serine
- Structural and mechanistic insights into the inhibition of class C beta-lactamases through the adenylylation of the nucleophilic serine
- Kim, Min-Kyu; An, Young Jun; Na, Jung-Hyun; Seol, Jae-Hee; Ryu, Ju Yeon; Lee, Jin-Won; Kang, Lin-Woo; Chung, Kyung Min; Lee, Jung-Hyun; Moon, Jeong Hee; Lee, Jong Seok; Cha, Sun-Shin
- Ewha Authors
- SCOPUS Author ID
- Issue Date
- Journal Title
- JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
- 0305-7453; 1460-2091
- vol. 72, no. 3, pp. 735 - 743
- OXFORD UNIV PRESS
- SCI; SCIE; SCOPUS
- Objectives: Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended spectrumclass C beta-lactamases. Methods: 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 50'-(P-acetyl) monophosphate ( acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates. Results: 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 bemodelled as a two-stepmechanism. 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. Conclusions: Our structural and kinetic studies revealed the detailed mechanismof adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C beta-lactamase inhibitors on the basis of the nucleotide scaffold.
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