Shaping Substrate Selectivity in a Broad-Spectrum Metallo-β-Lactamase

Abstract

Metallo-β-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature.

Keywords: SPM-1; beta-lactamases; mechanisms of resistance; metallo-beta-lactamase; substrate profile.

FIG 1

Design of SPM-1/Sfh-I. (A) Structural alignment of SPM-1 (black/red) and Sfh-I (gray/yellow) showing substitutions (1, L119T; 2, Y223N; 3, D226F) and swapping of sequence insertions to render SPM-1/Sfh-I. (B) Sequence swapping displayed on crystal structures of SPM-1 (PDB entry 2FHX) and Sfh-I (PDB entry 3SD9). (C) Representation of native interactions accommodating α3 insertion in Sfh-I. Zinc ions are depicted as purple spheres.

FIG 2

Western blot of spheroplasts and periplasmic fractions from P. aeruginosa PAO1 and E. coli DH5α, producers of SPM-1 or SPM-1/Sfh-I. Cells containing the empty vector (−) were used as a control. Anti-Strep-tag antibody was used to assess SPM-1 or SPM-1/Sfh-I production, while GroEL and MBP are spheroplasts and periplasm markers, respectively.

FIG 3

Folding and thermal stability of SPM-1 and SPM-1/Sfh-1. (A) Far-UV CD spectrum of wild-type SPM-1 (solid line) and SPM-1/Sfh-I hybrid protein (broken line). (B) Thermal denaturation of SPM-1 (solid line) and SPM-1/Sfh-I (broken line) followed by thermal shift (T_m of SPM-1/Sfh-I, 51°C ± 1°C; T_m of SPM-1, 75°C ± 0.5°C).

FIG 4

(A) Representative snapshots of the MD simulations of SPM-1^open (red), SPM-1/Sfh-I^open (violet), SPM-1/Sfh-I^closed (cyan), SPM-1^closed (pink), and Sfh-I (dark yellow), showing the accessibility of the active site. (B) Relative probability of association of the enzymes with PenG.