Metallo-beta-lactamases (MBLs) are members of the metallohydrolases family and constitute a very effective resistance mechanism employed by bacteria to escape the action of most beta-lactam antibiotics. Emergence of acquired MBLs among pathogenic species represents a major clinical threat, especially since no efficient inhibitors are available. On the basis of their primary structures, these enzymes have been subdivided in three subclasses (B1, B2 and B3).
The enzyme studied in this work, termed BcII, is produced by Bacillus cereus, strain 569/H/9, and is the most studied MBL so far (class B1 ; 227 a.a. ; M.W. 24960 Da). It displays a binuclear active site centre, which can bind various metal ions (e.g. Co2+ and Cd2+), although Zn2+ is the natural cofactor used for beta-lactams hydrolysis.
The first part of this work was dedicated to the characterization of the equilibrium folding properties of both the apo and holo forms of BcII. We used a variety of biophysical techniques, including absorbance, circular dichroism and intrinsic fluorescence spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS). Interestingly, optical measurements revealed that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its α-helical content, presumably associated with enhanced flexibility. The holoenzyme was found to be much more stable than the zinc-depleted form. Whereas the latter unfold according to a simple two-state mechanism, unfolding of the holoenzyme was found to be non-cooperative, with the population of intermediate species showing 3D structures very similar to the native species.
Besides folding studies, we investigated the process of metal binding to BcII. Zinc binding was monitored using complementary techniques, including circular dichroism in the far UV, enzymatic activity measurements, competition with a chromophoric chelator, MS and NMR. Most noticeably, MS and NMR experiments, together with catalytic activity measurements demonstrated that two zinc ions bind cooperatively to the enzyme active site (with K1/K2 ≥ 5, indicating positive cooperativity) and hence that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicated K2 < 80 nM.