The Quorum-Quenching Metallo-gamma-lactonase from Bacillus thuringiensis Exhibits a Leaving Group Thio Effect
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 {sup 18}O, {sup 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. {sup 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{sup 2+}, Co{sup 2+}, Zn{sup 2+}, and Cd{sup 2+}. The magnitude of the thio effect on k{sub 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{sup 2+} results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.
- Research Organization:
- Brookhaven National Laboratory (BNL) National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- AC02-98CH10886
- OSTI ID:
- 930153
- Report Number(s):
- BNL--80811-2008-JA
- Journal Information:
- Biochemistry, Journal Name: Biochemistry Vol. 45; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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