Quorum Sensing pp 275-290 | Cite as

Heterologous Overexpression, Purification, and In Vitro Characterization of AHL Lactonases

  • Pei W. Thomas
  • Walter Fast
Part of the Methods in Molecular Biology book series (MIMB, volume 692)


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 words

Quorum 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).


  1. 1.
    Dong, Y. H., Wang, L. H., and Zhang, L. H. (2007) Quorum-quenching microbial infections: mechanisms and implications, Philos. Trans. R. Soc. Lond. B Biol. Sci.362, 1201–1211.PubMedCrossRefGoogle Scholar
  2. 2.
    Kaufmann, G. F., Park, J., and Janda, K. D. (2008) Bacterial quorum sensing: a new target for anti-infective immunotherapy, Expert Opin. Biol. Ther. 8, 719–724.PubMedCrossRefGoogle Scholar
  3. 3.
    Kapadnis, P. B., Hall, E., Ramstedt, M., Galloway, W. R., Welch, M., and Spring, D. R. (2009) Towards quorum-quenching catalytic antibodies, Chem. Commun. (Camb.) 5, 538–540.CrossRefGoogle Scholar
  4. 4.
    Pustelny, C., Albers, A., Buldt-Karentzopoulos, K., Parschat, K., Chhabra, S. R., Camara, M., Williams, P., and Fetzner, S. (2009) Dioxygenase-mediated quenching of quinolone-dependent quorum sensing in Pseudomonas aeruginosa, Chem. Biol. 16, 1259–1267.PubMedCrossRefGoogle Scholar
  5. 5.
    Roy, V., Fernandes, R., Tsao, C. Y., and Bentley, W. E. (2010) Cross species quorum quenching using a native AI-2 processing enzyme, ACS Chem. Biol.5(2), 223–232.PubMedCrossRefGoogle Scholar
  6. 6.
    Xu, F., Byun, T., Deussen, H. J., and Duke, K. R. (2003) Degradation of N-acylhomoserine lactones, the bacterial quorum-sensing molecules, by acylase, J. Biotechnol. 101, 89–96.PubMedCrossRefGoogle Scholar
  7. 7.
    Park, S. Y., Kang, H. O., Jang, H. S., Lee, J. K., Koo, B. T., and Yum, D. Y. (2005) Identification of extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its application to quorum quenching, Appl. Environ. Microbiol. 71, 2632–2641.PubMedCrossRefGoogle Scholar
  8. 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
  9. 9.
    Momb, J., Thomas, P. W., Breece, R. M., Tierney, D. L., and Fast, W. (2006) The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect, Biochemistry 45, 13385–13393.PubMedCrossRefGoogle Scholar
  10. 10.
    De Lamo Marin, S., Xu, Y., Meijler, M. M., and Janda, K. D. (2007) Antibody catalyzed hydrolysis of a quorum sensing signal found in Gram-negative bacteria, Bioorg. Med. Chem. Lett. 17, 1549–1552.PubMedCrossRefGoogle Scholar
  11. 11.
    Chow, J. Y., Wu, L., and Yew, W. S. (2009) Directed evolution of a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the amidohydrolase superfamily, Biochemistry 48, 4344–4353.PubMedCrossRefGoogle Scholar
  12. 12.
    Dong, Y. H., Xu, J. L., Li, X. Z., and Zhang, L. H. (2000) AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U. S. A. 97, 3526–3531.PubMedCrossRefGoogle Scholar
  13. 13.
    Liu, D., Lepore, B. W., Petsko, G. A., Thomas, P. W., Stone, E. M., Fast, W., and Ringe, D. (2005) Three-dimensional structure of the quorum-quenching N-acyl homoserine lactone hydrolase from Bacillus thuringiensis, Proc. Natl. Acad. Sci. U. S. A. 102, 11882–11887.PubMedCrossRefGoogle Scholar
  14. 14.
    Liu, D., Momb, J., Thomas, P. W., Moulin, A., Petsko, G. A., Fast, W., and Ringe, D. (2008) Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures, Biochemistry 47, 7706–7714.PubMedCrossRefGoogle Scholar
  15. 15.
    Momb, J., Wang, C., Liu, D., Thomas, P. W., Petsko, G. A., Guo, H., Ringe, D., and Fast, W. (2008) On the mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis: 2. Substrate modeling and active site mutations, Biochemistry 47, 7715–7725. PubMedCrossRefGoogle Scholar
  16. 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
  17. 17.
    Wang, L. H., Weng, L. X., Dong, Y. H., and Zhang, L. H. (2004) Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase), J. Biol. Chem. 279, 13645–13651.PubMedCrossRefGoogle Scholar
  18. 18.
    Thomas, P. W., Stone, E. M., Costello, A. L., Tierney, D. L., and Fast, W. (2005) The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein, Biochemistry 44, 7559–7569.PubMedCrossRefGoogle Scholar
  19. 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
  20. 20.
    Kristelly, R., Earnest, B. T., Krishnamoorthy, L., and Tesmer, J. J. (2003) Preliminary structure analysis of the DH/PH domains of leukemia-associated RhoGEF, Acta. Crystallogr. D Biol. Crystallogr. 59, 1859–1862.PubMedCrossRefGoogle Scholar
  21. 21.
    Kapust, R. B., Tozser, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency, Protein Eng. 14, 993–1000.PubMedCrossRefGoogle Scholar
  22. 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
  23. 23.
    Khalifah, R. G. (1971) The carbon dioxide hydration activity of carbonic anhydrase. I. Stop–flow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246, 2561–2573.PubMedGoogle Scholar
  24. 24.
    Hurt, J. D., Tu, C., Laipis, P. J., and Silverman, D. N. (1997) Catalytic properties of murine carbonic anhydrase IV, J. Biol. Chem. 272, 13512–13518.PubMedCrossRefGoogle Scholar
  25. 25.
    Schindler, J. F., Naranjo, P. A., Honaberger, D. A., Chang, C. H., Brainard, J. R., Vanderberg, L. A., and Unkefer, C. J. (1999) Haloalkane dehalogenases: steady-state kinetics and halide inhibition, Biochemistry 38, 5772–5778.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Pei W. Thomas
    • 1
  • Walter Fast
    • 1
  1. 1.Division of Medicinal Chemistry, College of PharmacyUniversity of TexasAustinUSA

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