Abstract/3-Lactamases are extracellular or periplasmic bacterial enzymes which confer resistance to/3-1actam antibiotics. On the basis of their catalytic mechanisms, they can be divided into two major groups: active-site serine enzymes (classes A, C and D) and the Zn II enzymes (class B). The first crystal structure of a class B enzyme, the metallo-fllactamase from Bacillus cereus, has been solved at 2.5 A resolution [Carfi, Pares, Du6e, Galleni, Duez, Fr~re & Dideberg (1995). EMBO J. 14, 4914---4921]. Recently, the crystal structure of the metallo-/3-1actamase from Bacteroides fragilis has been determined in a tetragonal space group [Concha, Rasmussen, Bush & Herzberg (1996). Structure, 4, 823-836]. The structure of the metallo-/3-1actamase from B. fragilis in an orthorhombic crystal form at 2.0 A resolution is reported here. The final crystallographic R is 0.196 for all the 32 501 observed reflections in the range 10-2.0 A. The refined model includes 458 residues, 437 water molecules, four zinc and two sodium ions. These structures are discussed with reference to Zn binding and activity. A catalytic mechanism is proposed which is coherent with metallo-/~-lactamases being active with either one Zn ion (as in Aeromonas hydrophila) or two Zn ions (as in B. fragilis) bound to the protein.

When expressed by pathogenic bacteria, Zn2؉ -␤-lactamases induce resistance to most ␤-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn 2؉ -␤-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn 2؉ -␤-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn 2؉ (mononuclear) and di-Zn 2؉ (binuclear) Zn 2؉ -␤-lactamases are catalytically active but with different kinetic properties. The mono-Zn2؉ -␤-lactamase requires the conserved cysteine residue for hydrolysis of the ␤-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.

Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectroscopy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn(2)E, ZnE, Cd(2)E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.