Heterologous Overexpression, Purification, and In Vitro Characterization of AHL Lactonases
Quorum-quenching enzymes are useful as biochemical tools and possible therapeutic proteins. One of the best-characterized families of these catalysts is the N-acyl-l-homoserine lactone (AHL) lactonases, which rely on a dinuclear metal ion active site to hydrolytically cleave the autoinducer’s lactone bond and inactivate signaling. A detailed understanding of how this enzyme works can help in the design of more selective and efficient reagents. To facilitate these studies, we describe a methodology to heterologously express, purify, and conduct in vitro characterization of several metalloforms of the AHL lactonase from Bacillus thuringiensis (AiiA). These procedures should be applicable to similar enzymes and will facilitate the production of more useful quorum-quenching reagents for biochemical studies and possible therapeutic applications.
Key wordsQuorum quenching Lactonase N-acyl-l-homoserine lactone Protein purification Metalloprotein Enzyme kinetics
This work was supported in part by the Texas Advanced Research Program (Grant 003658-0018-2006) and the Robert A. Welch Foundation (Grant F-1572).
- 8.Teiber, J. F., Horke, S., Haines, D. C., Chowdhary, P. K., Xiao, J., Kramer, G. L., Haley, R. W., and Draganov, D. I. (2008) Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone, Infect. Immun. 76, 2512–2519.PubMedCrossRefGoogle Scholar
- 16.Kim, M. H., Choi, W. C., Kang, H. O., Lee, J. S., Kang, B. S., Kim, K. J., Derewenda, Z. S., Oh, T. K., Lee, C. H., and Lee, J. K. (2005) The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-l-homoserine lactone hydrolase, Proc. Natl. Acad. Sci. U. S. A. 102, 17606–17611.PubMedCrossRefGoogle Scholar
- 19.Sambrook, J., and Russell, D. W. (2001) Molecular cloning : a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
- 22.Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1960) The role of zinc in carboxypeptidase, J. Biol. Chem. 235, 64–69.Google Scholar