The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase

Abstract

In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-angstroms resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-angstroms crystal structure of its complex with L-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-beta-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases.

Fig. 1.

Overall structure of BTK-AiiA and structural comparison of various zinc-metalloenzymes. (A) Ribbon representation of BTK-AiiA. α helices and β sheets are shown in purple and green, respectively. The flexible fragment of disordered electron density shown in red was stereochemically modeled by using the program o. (B) Human glyoxalase II (PDB ID code 1QH5) and (C) RNase Z (PDB ID code 1Y44) are shown. All structures are represented with two zinc ions as lime spheres in active sites. rms deviation values are 2.6 and 1.6 Å for human glyoxalase II (160 superimposed α-carbons) and RNase Z (96 super-imposed α-carbons), respectively, compared with BTK-AiiA. (D) Superposition of the C-terminal BTK-AiiA domain in green onto the N-terminal domain in purple, colored according to temperature factors, where thicker represents higher B values. rms deviation is 1.34 Å for the C-terminal domain (34 super-imposed α-carbons) compared with the N-terminal domain. Red loop indicates flexible fragment in BTK-AiiA. (E) Metal-binding center in BTK-AiiA. Interactions for metal coordination are outlined by black long-dashed lines, and hydrogen bonds are depicted as gray short-dashed lines. Interactions were measured in angstroms. Carbons, oxygens, and nitrogens are shown in green, red, and blue, respectively. Water molecules are indicated as red spheres, and zinc ions are shown as gray spheres. Unless otherwise noted, figures were prepared by using pymol (DeLano Scientific, South San Francisco, CA).

Fig. 2.

Mutagenic analysis of functional residues in the metal-binding center. (A) The assay was performed in the presence of 0.1 mM ZnCl₂ or 0.1 mM N,N,N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine. Enzyme activity was analyzed by LC-ESI-MS. (B) A bioassay of enzymes was performed. Residual amounts of C6-AHL were evaluated according to the decrease in size of purple-colored areas around the hole in the CV026 plate. (C) The zinc contents of BTK-AiiA and its mutants. The amounts of zinc in tag-free BTK-AiiA (WT), MBP-6×His-fusion WT, and the mutant enzymes were determined by inductively coupled plasma atomic emission spectrometry, as described in Supporting Text.

Fig. 3.

Substrate specificity of BTK-AiiA and inhibition of BTK-AiiA activity by HSL. (A) Activity toward C6-AHL (68.6 nmol/min per mg) was defined as 100%. (B) A typical steady-state kinetics experiment of BTK-AiiA activity for different C6-AHL concentrations was performed at increasing concentrations of l-HSL. Enzyme activity was analyzed by LC-ESI-MS, as described in Supporting Text.

Fig. 4.

Substrate binding by BTK-AiiA. (A) Electron density map showing the bound l-HSL in the active site of BTK-AiiA. The F_o–F_c map was calculated before the inclusion of l-HSL in the model and is contoured at 2.5 σ. (B) HSL-binding site and hydrophobic channel in BTK-AiiA. The hydrophobic channel is accommodated with several hydrophobic side chains and extended from the HSL-binding site. BTK-AiiA-HSL features are shown in purple, and those of the native BTK-AiiA are shown in green. HSL is shown in yellow, and zinc ions are indicated as gray spheres. (C) Model of AHL-binding in BTK-AiiA. The model was generated based on modeled HSL binding in BTK-AiiA by using the program o. The disordered flexible region in red was stereochemically modeled by using the program o.

Fig. 5.

Proposed catalysis mechanism for BTK-AiiA based on the BTK-AiiA–HSL complex and suggested catalysis mechanisms for other zinc metalloenzymes. See text for description.