Structural and mechanistic insights into the inhibition of class C β-lactamases through the adenylylation of the nucleophilic serine

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

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7 Scopus citations

Abstract

Objectives: Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C β-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 5′-(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 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 (kinact/KI) 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 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.

Original languageEnglish
Pages (from-to)735-743
Number of pages9
JournalJournal of Antimicrobial Chemotherapy
Volume72
Issue number3
DOIs
StatePublished - 1 Mar 2017

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