Mutagenesis and Steady State Kinetic Characterization of a Class B Metallo‐β‐lactamase

Antibiotic resistance is of growing concern for the medical community worldwide due to the overuse of common antibiotics that has created an opportunity for bacteria to evolve novel mechanisms for resistance to antibiotics. Specifically, bacteria that express enzymes that inactivate β‐lactams prevent the antibiotics from inhibiting cell wall biosynthesis in bacteria. Class B β‐lactamases, metallo‐β‐lactamases, are of particular concern. These metal dependent enzymes are resistant to almost all classes of β‐lactams, including carbapenems, which have the broadest spectrum of activity. One such enzyme is β‐lactamase 2 [Bc(II)] from Bacillus cereus , a mono‐ or di‐zinc dependent metallo‐β‐lactamase. The amide bond hydrolysis of β‐lactams by Bc(II) is likely facilitated by a zinc(II)‐bound water and the amino acid residue aspartate‐90, which is thought to participate in general acid base catalysis. Bicknell and coworkers suggest that the metal acts as a Lewis Acid and binds a hydroxide ion that carries out a direct nucleophilic attack on the β‐lactam carbonyl. The metal is suggested to stabilize the resulting tetrahedral intermediate, allowing for the carboxylate anion of aspartate‐90 to deprotonate the metal‐bound hydroxide and then protonate the amine nitrogen. To more thoroughly understand the role of aspartate‐90, the steady state kinetics of wildtype, aspartate‐90‐asparagine, and aspartate‐90‐alanine mutant are being evaluated.