The directed evolution of NDM-1

Abstract

β-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward β-lactam antibiotics is the production of β-lactamases. These enzymes are capable of hydrolyzing the β-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- β-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- β-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 β-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

Keywords: NDM-1; antibiotic resistance; antimicrobial resistance; beta-lactamases; beta-lactams; drug resistance evolution.

Fig 1

Immunoblot for NDM-1 WT, NDM-9, and six mutants generated by error-prone PCR. The anti-NDM antibody used is derived from mouse serum.

Fig 2

Representations of the mutations made to wild-type NDM-1. (A) The wild-type NDM-1 structure was taken from PDB: 4EXY37. The gray spheres represent the two active site zinc ions. Active site residues coordinating the zinc ions are shown as white sticks. The T173S mutation (variant 3-9) is shown in cyan. The E152K mutation (variant 5-14) is shown in yellow. The magenta color for residue 42, the first residue present in the crystal structure, shows the location of the amino-terminus where the E2D mutation of mutant 5-14 would be located (the first ~40 amino acid residues are not present in crystal structures). (B) Close-up view of the E152K mutation shown with an AlphaFold model of NDM-1 E152K (dark gray) superimposed onto the wild-type NDM-1 structure (light gray). Active site zinc ions in the background are shown as spheres. Electrostatic interactions between Lys152 and Asp223 are shown as dashed lines (yellow) with labeled distances in Å. (C) Close-up view of the T173S mutation shown with an AlphaFold model of NDM-1 T173S (dark gray) superimposed onto the wild-type NDM-1 structure (light gray).