Please use this identifier to cite or link to this item: http://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, 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
RMID: 0030071036
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
Appears in Collections:Chemistry publications

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