Fig. 1.

Active site residues of metallo-β-lactamases. a Sequence alignment of representative metallo-β-lactamases from subclasses B1, B2, and B3. The amino acids highlighted in blue and yellow indicate the histidine and cysteine zinc binding site residues, respectively. NDM-1 residues are in bold. b Schematic representation of β-lactam substrate binding (penicillin), anionic intermediate stabilization, and product release in the active site of di-zinc metallo-β-lactamase NDM-1. The substrate binds to the active site through interaction of the carbonyl oxygen of the β-lactam ring with Zn1 and the carboxyl group on the fused ring with Zn2 and residue Lys224. A hydroxide ion stabilized by Zn1 and Zn2 attacks the carbonyl carbon of the β-lactam ring, leading to the formation of a carboxylate group and a nitrogen anion. The former is coordinated by Zn1 and the side chain of the conserved Asn233. The latter is stabilized by Zn2 and protonated coincident with or after C–N bond cleavage. The proton donor for the anionic nitrogen is shown as the newly formed carboxylate, however, the proton has also been proposed to be donated by a water. c Diagram of the NDM-1 β-lactamase structure highlighting active site residues for which random mutant libraries were created (magenta). The zinc atoms are represented as orange spheres. The figure was rendered with using coordinates from the Protein Data Bank accession code 3SPU¹⁸