The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect

Abstract

Lactone-hydrolyzing enzymes derived from some Bacillus species are capable of disrupting quorum sensing in bacteria that use N-acyl-l-homoserine lactones (AHLs) as intercellular signaling molecules. Despite the promise of these quorum-quenching enzymes as therapeutic and anti-biofouling agents, the ring opening mechanism and the role of metal ions in catalysis have not been elucidated. Labeling studies using (18)O, (2)H, and the AHL lactonase from Bacillus thuringiensis implicate an addition-elimination pathway for ring opening in which a solvent-derived oxygen is incorporated into the product carboxylate, identifying the alcohol as the leaving group. (1)H NMR is used to show that metal binding is required to maintain proper folding. A thio effect is measured for hydrolysis of N-hexanoyl-l-homoserine lactone and the corresponding thiolactone by AHL lactonase disubstituted with alternative metal ions, including Mn(2+), Co(2+), Zn(2+), and Cd(2+). The magnitude of the thio effect on k(cat) values and the thiophilicity of the metal ion substitutions vary in parallel and are consistent with a kinetically significant interaction between the leaving group and the active site metal center during turnover. X-ray absorption spectroscopy confirms that dicobalt substitution does not result in large structural perturbations at the active site. Finally, substitution of the dinuclear metal site with Cd(2+) results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.

Scheme 1

Enzymic hydrolysis of quorum-sensing AHL signals by a) AHL acylases, which target the amide bond for hydrolysis or b) paroxonases and AHL lactonases, which target the lactone bond for hydrolysis. Either pathway yields inactive products.

Scheme 2

Possible AHL ring-opening mechanisms by elimination of the acetate (a) possibly followed by hydration (a′); by hydroxide attack at the Cγ-carbon (b); by elimination of the alcohol (c) followed by hydration (c′); or by hydroxide attack at the carbonyl carbon (d). Predicted labeling by solvent [¹⁸O]-H₂O is indicated (●).

Figure 1

Partial ¹³C NMR spectra of ¹⁸O-labeled product. C6-HSL was hydrolyzed by AHL lactonase in 50 % H₂¹⁸O / 50 % H₂¹⁶O, and two selected regions of the ¹³C NMR spectrum of the resulting product, N-hexanoyl-L-homoserine, are shown, corresponding to the carboxylic carbon (approx 179 ppm) and the Cγ methylene carbon (approx 72 ppm).

Figure 2

Partial one-dimensional ¹H NMR spectra of dinuclear zinc AHL lactonase (A) and apo AHL lactonase (B), indicating significant unfolding after removal of metal ions.

Figure 3

Fourier transformed EXAFS data (solid lines) for dizinc (top) (19) and dicobalt (bottom) AHL lactonase, and corresponding best fits (open diamonds).

Figure 4

Proposed metal-to-leaving-group interaction during lactone and thiolactone hydrolysis by AHL lactonase.