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. 2016 Jan;12(1):9-14.
doi: 10.1038/nchembio.1950. Epub 2015 Nov 9.

Class D β-lactamases do exist in Gram-positive bacteria

Marta Toth  1 , Nuno Tiago Antunes  1 , Nichole K Stewart  1 , Hilary Frase  1 , Monolekha Bhattacharya  1 , Clyde A Smith  2 , Sergei B Vakulenko  1
Affiliations

Class D β-lactamases do exist in Gram-positive bacteria

Marta Toth et al. Nat Chem Biol. 2016 Jan.

Abstract

Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. These enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.

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Figures

Figure 1
Figure 1. The BPU-1 structure
(a) Ribbon representation of the BPU-1 crystal structure. The secondary structure assignment is indicated. The sidechain of the catalytic serine residue, Ser101, indicates the location of the enzyme active site. Single letter amino acid abbreviations are used throughout the Figures for clarity. (b) Final 2Fo-Fc electron density near the BPU-1 active site, contoured at 1.2σ. Electron density for the carboxylate moiety covalently attached to the side chain of Lys104 is evident. The carboxylated lysine is anchored by two hydrogen bonding interactions to a highly-conserved tryptophan residue (Trp188) from a loop adjacent to the active site. This loop is in turn anchored to the N-terminus of helix α3 by an additional hydrogen bonding interaction. (c) Superposition of BPU-1 (gray ribbons) and OXA-23 (cyan ribbons). Only strands equivalent to β8, β9, and β10, along with helix α9, are shown for OXA-23 for clarity. Two loops (Loop1 and Loop2) which show the largest structural deviation between BPU-1 and the OXA enzymes are shown in red for BPU-1 and blue for OXA-23.
Figure 2
Figure 2. Structure-based sequence alignment of Gram-positive class D β-lactamases
A segment of the sequence alignment of BPU-1 and other class D β-lactamase enzymes identified in various Bacillus species is shown, covering the two fingerprint loops (Loop1 and Loop2, highlighted in light gray) which show the largest structural variation compared to the Gram-negative OXA enzymes. OXA-10, OXA-23 and OXA-24 are shown as the representative Gram-negative β-lactamases. The secondary structure assignment for BPU-1 is indicated at the top. The conserved arginine residue in the Gram-negative enzymes is highlighted green, and the equivalent alanine residue is highlighted yellow. Residues and motifs which are highly conserved in the Gram-negative and Gram-positive enzymes are colored red.
Figure 3
Figure 3. BPU-1 substrate binding
(a) Close up view of the arginine pocket in OXA-23 (cyan ribbons, blue loop and lighter blue sticks), superimposed on the equivalent region near Loop 2 in BPU-1 (gray ribbons and red loop). The conserved Gram-negative class D arginine residue (represented by Arg259 from OXA-23) responsible for anchoring the carboxylate group of meropenem (cyan ball-and-stick) in OXA-23 is indicated. (b) The general structure of the carbapenem antibiotics. Differences in the structure of the tail group (R) distinguish the various clinically-available antibiotics including imipenem, meropenem, doripenem, and ertapenem. The R’ position is occupied either by hydrogen in imipenem) or by a methyl group in meropenem, doripenem, and ertapenem. (c) The substrate binding site in the doripenem-BPU-1 complex. The BPU-1 structure is represented by green ribbons and sticks and the covalently-linked doripenem by yellow sticks. The hydrogen bonding interactions between the doripenem and the protein are shown as dashed black lines. (d) The difference in coordination of doripenem (yellow sticks) in BPU-1 (grey ribbons, grey sticks) and meropenem (cyan sticks) in OXA-23. Only the residues which interact with the meropenem are shown for OXA-23 (light blue sticks) for clarity, and only the hydrogen bonding in the meropenem-OXA-23 complex is shown. The OXA-23 residues are labeled in italics and the BPU-1 residues in plain bold text. The tail of the doripenem in BPU-1 is truncated at the sulfur atom for clarity.
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