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. 2016 Sep;72(Pt 9):681-6.
doi: 10.1107/S2053230X16011821. Epub 2016 Aug 9.

The quorum-quenching lactonase from Geobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis

Celine Bergonzi  1 , Michael Schwab  1 , Mikael Elias  1
Affiliations

The quorum-quenching lactonase from Geobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis

Celine Bergonzi et al. Acta Crystallogr F Struct Biol Commun. 2016 Sep.

Abstract

Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-β-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacterium Geobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidate for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75°C of 152.5 ± 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (kcat/Km) against AHLs of greater than 10(6) M(-1) s(-1). The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 Å resolution of GcL are reported.

Keywords: lactonase; quorum quenching; quorum sensing; thermophile.

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Figures

Figure 1
Figure 1
Putative catalytic mechanism of lactonases in the metallo-β-lactamase family. Adapted from Liu et al. (2008 ▸) and Momb et al. (2008 ▸).
Figure 2
Figure 2
12% SDS–PAGE of the GcL protein. Lane M contains molecular-weight markers (Precision Plus Protein Kaleidoscope Prestained Protein Standards, Bio-Rad; labelled in kDa). Lane 1 contains 5 µl GcL protein at 1 mg ml−1.
Figure 3
Figure 3
The half-life of GcL incubated at 70 and 75°C. The enzyme was incubated for different times at two different temperatures and the lactonase activity against C6-AHL is reported. The inferred half life (t 1/2) of the protein is 152.5 ± 10 and 109.1 ± 7 min at 70 and 75°C, respectively.
Figure 4
Figure 4
Crystals of the GcL protein appeared after 1 d at 20°C.
Figure 5
Figure 5
A diffraction frame from a crystal of GcL. The edge of the frame is at 1.40 Å resolution.
See this image and copyright information in PMC

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