The quorum-quenching lactonase from Alicyclobacter acidoterrestris: purification, kinetic characterization, crystallization and crystallographic analysis

Abstract

Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo-β-lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacterium Alicyclobacter acidoterrestris. Kinetic characterization proves AaL to be a proficient lactonase, with catalytic efficiencies (k_cat/K_m) against AHLs in the region of 10⁵ M^-1 s^-1. AaL exhibits a very broad substrate specificity. Its structure is expected to reveal the molecular determinants for its substrate binding and specificity, as well as to provide grounds for future protein-engineering projects. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection of AaL at 1.65 Å resolution are reported.

Keywords: Alicyclobacter acidoterrestris; lactonases; quorum quenching; quorum sensing; thermophiles.

Figure 1

12% SDS–PAGE of the AaL protein. Lane M contains molecular-weight markers (Precision Plus Protein Kaleidoscope Prestained Protein Standards from Bio-Rad; labelled in kDa). Lane 1 contains 10 µl AaL protein at 2.5 mg ml⁻¹. The black arrow indicates the band for AaL.

Figure 2

Thermal stability determination of AaL using ANS as a fluorescent probe.

Figure 3

Typical crystals of the AaL protein. 1 scale unit = 110 µm.

Figure 4

A diffraction frame from a crystal of AaL. The edge of the frame is at 1.23 Å resolution.