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|Title:||Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents|
|Author:||Monteiro Pedroso, M.|
|Citation:||Dalton Transactions, 2017; 46(39):13194-13201|
|Publisher:||Royal Society of Chemistry|
|Marcelo Monteiro Pedroso, Christopher Selleck, Jessica Bilyj, Jeffrey R. Harmer, Lawrence R. Gahan, Nataša Mitić, Alistair J. Standish, David L. Tierney, James A. Larrabeef and Gerhard Schenk|
|Abstract:||CpsB is a metal ion-dependent hydrolase involved in the biosynthesis of capsular polysaccharides in bacterial organisms. The enzyme has been proposed as a promising target for novel chemotherapeutics to combat antibiotic resistance. The crystal structure of CpsB indicated the presence of as many as three closely spaced metal ions, modelled as Mn(2+), in the active site. While the preferred metal ion composition in vivo is obscure Mn(2+) and Co(2+) have been demonstrated to be most effective in reconstituting activity. Using isothermal titration calorimetry (ITC) we have demonstrated that, in contrast to the crystal structure, only two Mn(2+) or Co(2+) ions bind to a monomer of CpsB. This observation is in agreement with magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) data that indicate the presence of two weakly ferromagnetically coupled Co(2+) ions in the active site of catalytically active CpsB. While CpsB is known to be a phosphoesterase we have also been able to demonstrate that this enzyme is efficient in hydrolyzing the β-lactam substrate nitrocefin. Steady-state and stopped-flow kinetics measurements further indicated that phosphoesters and nitrocefin undergo catalysis in a conserved manner with a metal ion-bridging hydroxide acting as a nucleophile. Thus, the combined physicochemical studies demonstrate that CpsB is a novel member of the dinuclear metallohydrolase family.|
|Keywords:||Streptococcus pneumoniae; Cobalt; Manganese; Cephalosporins; Bacterial Proteins; Anti-Infective Agents; Calorimetry; Crystallography, X-Ray; Circular Dichroism; Electron Spin Resonance Spectroscopy; Binding Sites; Catalytic Domain; Hydrolysis; Kinetics; Protein Tyrosine Phosphatases; Biocatalysis|
|Rights:||This journal is © The Royal Society of Chemistry 2017|
|Appears in Collections:||Chemistry publications|
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