Active site loops of membrane-anchored metallo-β-lactamases from environmental bacteria determine cephalosporinase activity

Abstract

Antimicrobial resistance is a significant global public health threat that limits treatment options for bacterial infections. This situation is aggravated by the environmental spread of β-lactamase genes. In particular, metallo-β-lactamases (MBLs) hydrolyze almost all available β-lactam antibiotics, including late-generation cephalosporins and carbapenems. Among MBLs, the New Delhi metallo-β-lactamase (NDM-1) of subclass B1 has shown the most ominous dissemination. NDM variants are the only MBLs of clinical importance that are membrane-anchored, a sub-cellular localization that endows them with high stability under conditions of metal limitation. However, antibiotic resistance predates modern antibiotic usage, and environmental bacteria serve as reservoirs for resistance genes. Here, we report the biochemical and structural characterization of two membrane-bound MBLs: CJO-1 and CIM-2, from Chryseobacterium joostei and Chryseobacterium indologenes, respectively. Both MBLs confer β-lactam resistance on producer bacterial strains and hydrolyze several antibiotics, although with impaired efficiency compared to NDM-1. Crystal structures reveal differences, compared to previously studied B1 MBLs, in the active site loops and their dynamic properties that impact activity. Specifically, a hindered access to the active site with the contribution of a Tyr residue in loop L10 and the presence of a positively charged Lys residue in loop L3 limit hydrolysis of cephalosporins with charged C3 substituents. Some of these novel features are preserved in other MBLs from Chryseobacterium spp. These findings suggest that Chryseobacterium spp. could act as reservoirs of MBL genes, while informing on the diversity of structure-function relationships and dynamic behaviors within the B1 subclass of these enzymes.

Keywords: Chryseobacterium spp.; NDM; membrane-anchoring; metallo-beta-lactamase.

Fig 1

Protein sequence alignment of CJO-1 and CIM-2 with NDM-1, IMP-1, VIM-2, and other Chryseobacterium B1 MBLs. The consensus Zn(II) ligands are indicated with an asterisk (*), putative lipobox sequences are underlined, and lipoboxes Cys are highlighted in yellow. The numbers indicate the residue positions at the ends of the sequences which differ from the standard numbering (BBL numbering [28, 29]). Loop L3 is indicated by a horizontal blue bar and corresponds to residues 59–67 in the standard numbering; loop L10 is indicated by a horizontal red bar and corresponds to residues 223–241 in the standard numbering. Dashes indicate gaps to align the sequences. The sequence alignment was visualized with Jalview (30).

Fig 2

Immunodetection of the putative Chryseobacterium MBLs CJO-1 (A) and CIM-2 (B) expressed in Escherichia coli. The lipoprotein NDM-1 is included as a positive control of a membrane-bound MBL. WC, whole cells; S, soluble fraction of the lysate; M, total membranes of the lysate; MM, molecular marker. The bands between 25 and 30 kDa correspond to the expressed MBLs. The band at 60 kDa corresponds to GroEL, used as a control.

Fig 3

Crystal structures of CJO-1 and CIM-2. Zn coordination residues are represented by sticks, Zn ions by gray spheres, and water molecules by red spheres. X-ray structure of CJO-1 (green, PDB 9GX9) from C. joostei (A). X-ray structure of CIM-2 (orange, PDB 9GX8) from C. indologenes (B). Superimposition of CJO-1 (green), CIM-2 (orange), and NDM-1 (cyan, PDB 5ZGY), showing the positions of key active site loops and the extended NDM-1 loop at position 169–192 (C).

Fig 4

Active site loops in CJO-1, CIM-2, and NDM-1. CJO-1 is colored green, CIM-2 orange, and NDM-1 cyan. Zn coordination residues and significant substitutions are presented as sticks; Zn ions (Zn1 and Zn2) by gray spheres and water molecules by red spheres. Superimpositions of CJO-1, CIM-2, and NDM-1, with loops L3 and L10 colored according to the protein (A). Loop L10 conformations in CJO-1, CIM-2, and NDM-1, with active site residues involved in ligand binding highlighted (B). Loop L3 conformations in CJO-1, CIM-2, and NDM-1, with significant amino acids around the active site labeled (note, including at position 87 which is not on loop L3, but is potentially involved in ligand binding) and colored according to their respective proteins (C).

Fig 5

Representative structures obtained from molecular dynamics simulations. CJO-1 (A) and NDM-1 (B) are shown as cartoon representations with the catalytic Zn(II) ions and the oxygen atom of the catalytic hydroxyl ion shown as gray and red spheres, respectively. The electrostatic potential surfaces of CJO-1 (C) and NDM-1 (D) with negative, neutral, and positive potentials colored as red, white, and blue, respectively, in a scale from −8 to 8 k_BT/e_c.