Gram-negative bacteria use N-acyl homoserine lactones (AHLs) to sense population density and regulate gene expression, including virulent phenotypes. The quorum-quenching AHL lactonase from Bacillus thuringiensis cleaves the lactone ring of AHLs, disabling this mode of gene regulation. Despite the potential applications of this enzyme as an antibacterial weapon, little was known about it's lactone ring-opening mechanism. As a member of the metallo-beta-lactamase superfamily, AHL lactonase requires two divalent metal ions for catalysis. NMR experiments confirm that these metal ions are also involved in proper enzyme folding. The chemical mechanism of ring opening was explored using isotope incorporation studies, and hydrolysis was determined to proceed via a nucleophilic attack by a solvent-derived hydroxide at the carbonyl of the lactone ring. A transient, kinetically significant metal-leaving group interaction was detected in steady-state kinetic assays with AHL lactonase containing alternative divalent metal ions hydrolyzing a sulfur-containing substrate. High-resolution crystal structures implicated two residues in substrate binding and hydrolysis, Tyr194 and Asp108. Site-directed mutagenesis of these residues followed by steady-state kinetic studies with wild-type and mutant enzymes hydrolyzing a spectrum of AHL substrates revealed that mutations Y194F and D108N significantly affect catalysis. Combining these results allows the proposal of a detailed hydrolytic mechanism. The binding site for the N-acyl hydrophobic moiety was probed using steady-state kinetics with a variety of naturally occurring and non-natural AHL substrates, and these studies indicate that AHL lactonase will accept a broad range of homoserine lactone containing substrates. Crystal structures with AHL substrates and non-hydrolyzable analogs reveal two distinct binding sites for this N-acyl group. Based on the ability of this enzyme to accommodate a variety of substrates, AHL lactonase was shown to have the ability to quench quorum sensing regulated by a newly discovered class of homoserine lactone signal molecules possessing an N-aryl group using a bioassay. Steady-state kinetic studies confirm that this class of signal molecules are indeed substrates for AHL lactonase. / text

Yes / Bacteria sense their own population size, tune the expression of responding genes, and behave accordingly to environmental stimuli by secreting signaling molecules. This phenomenon is termed as quorum sensing (QS). By exogenously manipulating the signal transduction bacterial population behaviors could be controlled, which may be done through quorum quenching (QQ). QS related regulatory networks have been proven their involvement in regulating many virulence determinants in pathogenic bacteria in the course of infections. Interfering with QS signaling system could be a novel strategy against bacterial infections and therefore requires more understanding of their fundamental mechanisms. Here we review the development of studies specifically on the inhibition of production of N-acyl-homoserine lactone (AHL), a common proteobacterial QS signal. The opportunistic pathogen, Pseudomonas aeruginosa, equips the alkylquinolone (AQ)-mediated QS which also plays crucial roles in its pathogenicity. The studies in QQ targeting on AQ are also discussed. / University of Malaya High Impact Research Grants (UMC/625/1/HIR/MOHE/CHAN/01, A-000001-50001,and UMC/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Yes / Burkholderia spp. rely on N-acyl homoserine lactone as quorum-sensing signal molecules which coordinate their phenotype at the population level. In this work, we present the whole genome of Burkholderia sp. strain A9, which enables the discovery of its N-acyl homoserine lactone synthase gene. / UM High Impact Research Grants (UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/01, H-50001-A000001 and UMMOHE HIR Grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001- A000027)