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https://hdl.handle.net/2440/106851
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Type: | Journal article |
Title: | Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents |
Author: | Monteiro Pedroso, M. Selleck, C. Bilyj, J. Harmer, J. Gahan, L. Mitić, N. Standish, A. Tierney, D. Larrabee, J. Schenk, G. |
Citation: | Dalton Transactions: an international journal of inorganic chemistry, 2017; 46(39):13194-13201 |
Publisher: | Royal Society of Chemistry |
Issue Date: | 2017 |
ISSN: | 1477-9226 1477-9234 |
Statement of Responsibility: | 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 |
DOI: | 10.1039/c7dt01350g |
Grant ID: | http://purl.org/au-research/grants/nhmrc/1084778 http://purl.org/au-research/grants/arc/DP150104358 http://purl.org/au-research/grants/arc/FT120100694 |
Published version: | http://dx.doi.org/10.1039/c7dt01350g |
Appears in Collections: | Aurora harvest 8 Chemistry publications |
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