Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents

Monteiro Pedroso, Marcelo, Selleck, Christopher, Bilyj, Jessica, Harmer, Jeffrey R. , Gahan, Lawrence R. , Mitić, Natasa, Standish, Alistair J. , Tierney, David L. , Larrabee, James A. and Schenk, Gerhard (2017) Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents. Dalton Transactions, 46 39: 13194-13201. doi:10.1039/c7dt01350g


Author Monteiro Pedroso, Marcelo
Selleck, Christopher
Bilyj, Jessica
Harmer, Jeffrey R.
Gahan, Lawrence R.
Mitić, Natasa
Standish, Alistair J.
Tierney, David L.
Larrabee, James A.
Schenk, Gerhard
Title Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents
Formatted title
Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents
Journal name Dalton Transactions   Check publisher's open access policy
ISSN 1477-9234
1477-9226
Publication date 2017-05-26
Year available 2017
Sub-type Article (original research)
DOI 10.1039/c7dt01350g
Open Access Status Not yet assessed
Volume 46
Issue 39
Start page 13194
End page 13201
Total pages 1
Place of publication Cambridge, United Kingdom
Publisher Royal Society of Chemistry
Language eng
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 Mn2+, in the active site. While the preferred metal ion composition in vivo is obscure Mn2+ and Co2+ 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 Mn2+ or Co2+ 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 Co2+ 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 beta-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.
Formatted 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 Mn2+, in the active site. While the preferred metal ion composition in vivo is obscure Mn2+ and Co2+ 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 Mn2+ or Co2+ 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 Co2+ 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.
Keyword Metallo-Beta-Lactamase
Binuclear Metallohydrolases
Tyrosine Phosphatases
Bioremediator Glycerophosphodiesterase
Capsular Polysaccharide
Enterobacter-Aerogenes
Antibiotic-Resistance
Catalytic Mechanisms
Crystal-Structures
Binding
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID APP1084778
DP150104358
CHE1303852
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
School of Chemistry and Molecular Biosciences
Centre for Advanced Imaging Publications
 
Versions
Version Filter Type
Citation counts: Scopus Citation Count Cited 0 times in Scopus Article
Google Scholar Search Google Scholar
Created: Fri, 23 Jun 2017, 15:14:12 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences