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

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.

The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL

“…GcL (WP_017434252.1) is a lactonase isolated from the thermophilic bacteria Parageobacillus caldoxylosilyticus . GcL is a rare thermophilic representative within MLLs, with a half‐life of (152.5±10) min at 75 °C . Herein, we show that GcL is a proficient lactonase, which makes it a potent inhibitor of biofilm formation for the pathogen Acinetobacter baumannii that uses C6‐AHL for signaling .…”

“…A wide range of lactones were tested: C4‐AHL, C6‐AHL, C8‐AHL, C10‐AHL, 3‐oxo‐C8‐AHL, γ‐butyrolactone, γ‐heptalactone, γ‐nonalactone, γ‐decanolactone, δ‐valerolactone, δ‐octanolactone, δ‐nonalactone, δ‐decalactone, ϵ‐caprolactone, ϵ‐decalactone, and whiskey lactone. The kinetic parameters of GcL against C4‐, C6‐, C10‐, and 3‐oxo‐C8‐AHLs were previously published …”

“…MLLs possess a conserved dinuclear metal binding motif, HXHXDH, involved in the binding of two metal cations and exhibit an αβ/βα fold. Interestingly, kinetic characterization studies on MLLs suggest that they all exhibit a broad specificity spectrum, which is in contrast to the observed preference of PLLs for long‐chain AHLs. Despite this shared substrate preference, kinetic studies reveal differences: K M values vary dramatically from about the 1 m m range for some enzymes (e.g., AiiA, AiiB) to about 1 μ m range for others (e.g., AidC, AaL).…”

Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

“…For enzyme production, Escherichia coli BL21 (DE) 3 -carrying plasmids pGro7/GroEL for chaperones and pET22b with either SsoPox W263I, SsoPox 5A8 (V27G/P67Q/L72C/Y97S/ Y99A/T177D/R223L/L226Q/L228M/W263H) or N-terminal strep tagged GcL wild type were used (Bergonzi et al, 2016;Guendouze et al, 2017). E. coli E. cloni R 10G transformed with the plasmid pGEM R -3Zf(+) (constitutive expression of lacZ alpha peptide) was used in the virulence assay.…”

“…This lactonase was also shown to reduce mortality in a rat pneumonia model (Hraiech et al, 2014). The MLL GcL isolated from Parageobacillus caldoxylosilyticus which was recently characterized and exhibits broad AHL specificity was chosen as the second lactonase (Bergonzi et al, 2016(Bergonzi et al, , 2019Mahan et al, 2020). SsoPox W263I and GcL, which mainly differ in their ability to hydrolyze C 4 HSL, were used independently and in combination, to disrupt QS of P. aeruginosa PA14 and to investigate the role of lactonase specificity on phenotypes, gene expression and proteome regulation.…”