Abstract
Quorum quenching lactonases are enzymes that are capable of disrupting bacterial signaling based on acyl homoserine lactones (AHL) via their enzymatic degradation. In particular, lactonases have therefore been demonstrated to inhibit bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. Here we characterized biochemically and structurally a novel representative from the metallo-β-lactamase superfamily, named AaL that was isolated from the thermoacidophilic bacterium Alicyclobacillus acidoterrestris. AaL is a potent quorum quenching enzyme as demonstrated by its ability to inhibit the biofilm formation of Acinetobacter baumannii. Kinetic studies demonstrate that AaL is both a proficient and a broad spectrum enzyme, being capable of hydrolyzing a wide range of lactones with high rates (kcat/KM > 105 M−1.s−1). Additionally, AaL exhibits unusually low KM values, ranging from 10 to 80 µM. Analysis of AaL structures bound to phosphate, glycerol, and C6-AHL reveals a unique hydrophobic patch (W26, F87 and I237), involved in substrate binding, possibly accounting for the enzyme’s high specificity. Identifying the specificity determinants will aid the development of highly specific quorum quenching enzymes as potential therapeutics.
Highlights
Organism Alicyclobacillus acidoterrestris Bacillus thuringiensis Agrobacterium tumefaciens Chryseobacterium sp. strain StRB126 Muricauda olearia Mammals Mammals Sulfolobus solfataricus Vulcanisaeta moutnovskia Sulfolobus acidocaldarius Sulfolobus islandicus Pseudomonas aeruginosa
The metallo-β-lactamase like (MLLs) or autoinducer inactivator A (AiiA)-like represents another class of lactonases
We show that AaL is capable of degrading δ-lactones and γ-lactones with high catalytic proficiency (>104 M−1 s−1) and shows some weak, promiscuous phosphotriesterase activity on the insecticide-derivated paraoxon (Fig. 1)
Summary
Organism Alicyclobacillus acidoterrestris Bacillus thuringiensis Agrobacterium tumefaciens Chryseobacterium sp. strain StRB126 Muricauda olearia Mammals Mammals Sulfolobus solfataricus Vulcanisaeta moutnovskia Sulfolobus acidocaldarius Sulfolobus islandicus Pseudomonas aeruginosa. The most characterized enzyme from this family is the autoinducer inactivator A (AiiA) from Bacillus thuringiensis[26], but other representatives have been studied, such as AiiB27, AidC28, MomL29 or GcL30. MLLs, including AiiA, AidC, MomL, were reported to exhibit broad substrate range with respect to the acyl chain length of AHLs4, but their potential activity on δ-lactones or γ-lactones is unknown. KM values for AaL were found to range between 10 to 83 μM34 with AHLs as substrates, while several other MLLs were reported to exhibit much higher KM values (~1 mM for AiiA26,32, 440 μM for MomL29) with the exception of AidC (46–72 μM35). The crystal structure of AaL reveals a unique hydrophobic patch which could relate to its high specificity, and highlights a flexible active site loop which may be involved in substrate binding
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