The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase'. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column.

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.

Twenty ␤-lactam molecules, including penicillins, cephalosporins, penems, carbapenems, and monobactams, were investigated as potential substrates for Xanthomonas maltophilia ULA-511, Aeromonas hydrophila AE036, and Bacillus cereus 5/B/6 metallo-␤-lactamases. A detailed analysis of the kinetic parameters examined confirmed these enzymes to be broad-spectrum ␤-lactamases with different ranges of catalytic efficiency. Cefoxitin and moxalactam, substrates for the ␤-lactamases from X. maltophilia ULA-511 and B. cereus 5/B/6, behaved as inactivators of the A. hydrophila AE036 metallo-␤-lactamase, which appeared to be unique among the enzymes tested in this study. In addition, we report a new, faster, and reliable purification procedure for the B. cereus 5/B/6 metallo-␤-lactamase, cloned in Escherichia coli HB101.␤-Lactamases are bacterial hydrolases (EC 3.5.2.6) which exert their catalytic activity either by a reactive serine residue in the active site (active-site-serine ␤-lactamases) or by a divalent transition metal ion (most often Zn 2ϩ ) coordinated to histidine and/or cysteine residues (metallo-␤-lactamases) (1). These enzymes show two main characteristics: (i) rapid inactivation of carbapenem antibiotics and (ii) inhibition by EDTA, a chelator of divalent cations. Moreover, they are able to inactivate penicillins, cephalosporins, and serine-␤-lactamase inhibitors, such as 6-␤-iodopenicillanic acid and penicillanic acid sulfone (5). Their interaction with monobactams remains to be evaluated.The relevance of metallo-␤-lactamases in the clinical field is therefore of considerable importance. The Bacillus cereus metallo-␤-lactamase, BcII, was first reported as a rare curiosity, but other metallo-enzymes were discovered in other bacterial species, namely Bacteroides, Aeromonas, Pseudomonas, Xanthomonas, Flavobacterium, Legionella, and Serratia (for reviews, see references 14 and 16), most often isolated from human patients.Several gene sequences have subsequently been reported for these ␤-lactamases (12,15,17,19), demonstrating some degree of heterogeneity. The metallo-␤-lactamase from B. cereus is considered the prototype enzyme. Information concerning the mechanism of action has suggested that zinc ions are bound to a water molecule when the enzyme attacks the carbonyl group of the ␤-lactam nucleus of penicillins, cephalosporins, and carbapenems, while an aspartate residue (Asp-90) would appear to act as a general base to assist the hydrolysis of the amide bond (11).As previously reported (5), the enzymes from Aeromonas hydrophila AE036, B. cereus 5/B/6, and Xanthomonas maltophilia ULA-511 show a very broad spectrum of activity against penicillins, cephalosporins, and carbapenems. The sole exception is the metallo-␤-lactamase from A. hydrophila AE036, which is able to hydrolyze, with good efficiency, imipenem and, to a lesser degree, ampicillin. Moreover, 6-␤-iodopenicillanic acid and sulbactam (penicillanic acid sulfone) behave as substrates for all three enzymes, which is in contrast to their usual inhibitory pr...