title: Structural and mechanistic insights into the inhibition of class C β-lactamases through the adenylylation of the nucleophilic serine.
abstract: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C β-lactamases.
  
  
  
  : 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.
  
  
  
  : 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.
  
  
  
  : 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.
authors.affiliation: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST), Ansan, 15627, Republic of Korea.|Research Division for Biotechnology, Korea Atomic Energy Research Institute (KAERI), Jeongeup, 56212, Republic of Korea.
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST), Ansan, 15627, Republic of Korea.
authors.forename: Young Jun
authors.initials: YJ
authors.lastname: An
authors.affiliation: Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 03760, Republic of Korea.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Na
authors.affiliation: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST), Ansan, 15627, Republic of Korea.
authors.forename: Jae-Hee
authors.initials: JH
authors.lastname: Seol
authors.affiliation: Functional Genomics Research Center, Korea Research Institute Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
authors.forename: Ju Yeon
authors.initials: JY
authors.lastname: Ryu
authors.affiliation: Department of Life Science, Hanyang University, Seoul, 04763, Republic of Korea.
authors.forename: Jin-Won
authors.initials: JW
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul, 05029, Republic of Korea.
authors.forename: Lin-Woo
authors.initials: LW
authors.lastname: Kang
authors.affiliation: Department of Microbiology and Immunology, Chonbuk National University Medical School, Jeonju, 54896, Republic of Korea.
authors.forename: Kyung Min
authors.initials: KM
authors.lastname: Chung
authors.affiliation: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST), Ansan, 15627, Republic of Korea.|Marine Biotechnology, Korea University of Science and Technology (UST), Daejeon, 34113, Republic of Korea.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Functional Genomics Research Center, Korea Research Institute Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
authors.forename: Jeong Hee
authors.initials: JH
authors.lastname: Moon
authors.affiliation: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST), Ansan, 15627, Republic of Korea.|Marine Biotechnology, Korea University of Science and Technology (UST), Daejeon, 34113, Republic of Korea.
authors.forename: Jong Seok
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 03760, Republic of Korea.
authors.forename: Sun-Shin
authors.initials: SS
authors.lastname: Cha
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism; D002442:Ceftazidime / Q000494:pharmacology; D018360:Crystallography, X-Ray; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D012694:Serine / Q000737:chemistry*; D065093:beta-Lactamase Inhibitors / Q000494:pharmacology*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D065093:beta-Lactamase Inhibitors; D012694:Serine; D002442:Ceftazidime; D001618:beta-Lactamases