Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis

Biochemistry. 1996 Sep 17;35(37):12126-32. doi: 10.1021/bi960976h.


In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are highspin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximately 30 and an intense feature at 320 nm (epsilon approximately 1570 The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on kcat. These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteroides fragilis / enzymology*
  • Binding Sites
  • Cations, Divalent / metabolism
  • Cloning, Molecular
  • DNA Primers
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Metals / metabolism*
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Polymerase Chain Reaction
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Spectrophotometry, Atomic
  • Thermodynamics
  • beta-Lactamases / chemistry*
  • beta-Lactamases / isolation & purification
  • beta-Lactamases / metabolism*


  • Cations, Divalent
  • DNA Primers
  • Metals
  • Recombinant Proteins
  • beta-Lactamases