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- Structural and mechanistic insights into the inhibition of class C beta-lactamases through the adenylylation of the nucleophilic serine
- M K Kim; Y J An; J H Na; J H Seol; Ju Yeon Ryu; J W Lee; L W Kang; K M Chung; J H Lee; Jeong Hee Moon; J S Lee; S S Cha
- Bibliographic Citation
- Journal of Antimicrobial Chemotherapy, vol. 72, no. 3, pp. 735-743
- Publication Year
- 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 ( 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 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.
- Oxford Univ Press
- Appears in Collections:
- Division of Bio Technology Innovation > Core Research Facility & Analysis Center > 1. Journal Articles
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