Mimicking natural evolution in metallo-beta-lactamases through second-shell ligand mutations

Abstract

Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most beta-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.

Fig. 1.

Chemical structures of the β-lactam substrates used in this work. Side chains R1 and R2 are specified for cephalosporins.

Fig. 2.

Measured pseudo-first order rate constants of cefotaxime (A) and cephalexin (B) binding to WT BcII (filled circles) and M5 (open circles). Data fits are shown as solid lines. Enzyme concentrations were 1 μM (A) and 10 μM (B). The reactions were carried out in 10 mM Hepes (pH 7.5), 0.2 M NaCl, and 20 μM added Zn(II) at 6°C. The calculated K_s values were 37.5 and 4.3 μM (cefotaxime hydrolysis by BcII and M5, respectively) and 37 and 44 μM (cephalexin hydrolysis by BcII and M5, respectively). Errors were within ±10%.

Fig. 3.

Electronic spectra of Co(II)-substituted BcII (black line) and Co(II)-substituted M5 (gray line) in 10 mM Hepes (pH 7.5) and 0.2 M NaCl. The enzyme concentrations were 200 μM, and 3 equivalents of Co(II) were added.

Fig. 4.

Model of the BcII active site with the Gly-262 → Ser and Asn-70 → Ser mutations in the second coordination sphere of Zn(II).