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

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, in the active site. While the preferred metal ion composition in vivo is obscure Mn and Co 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 or Co 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 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.