Min-Kyu Kim1,2, Young Jun An1, Jung-Hyun Na3, Jae-Hee Seol1, Ju Yeon Ryu4, Jin-Won Lee5, Lin-Woo Kang6, Kyung Min Chung7, Jung-Hyun Lee1,8, Jeong Hee Moon4, Jong Seok Lee1,8, Sun-Shin Cha3. 1. Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST), Ansan, 15627, Republic of Korea. 2. Research Division for Biotechnology, Korea Atomic Energy Research Institute (KAERI), Jeongeup, 56212, Republic of Korea. 3. Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 03760, Republic of Korea. 4. Functional Genomics Research Center, Korea Research Institute Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea. 5. Department of Life Science, Hanyang University, Seoul, 04763, Republic of Korea. 6. Department of Biological Sciences, Konkuk University, Seoul, 05029, Republic of Korea. 7. Department of Microbiology and Immunology, Chonbuk National University Medical School, Jeonju, 54896, Republic of Korea. 8. Marine Biotechnology, Korea University of Science and Technology (UST), Daejeon, 34113, Republic of Korea.
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 ( 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.
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.