The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase

Abstract

In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-Å resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-Å crystal structure of its complex with L-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-␤-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases. quorum sensing ͉ lactonase ͉ metalloenzyme ͉ crystal structure

Functional Analysis of the Purified Anandamide-generating Phospholipase D as a Member of the Metallo-β-lactamase Family

“…Single mutation of the residues corresponding to His-185, His-187, Asp-189, and His-253 of NAPE-PLD always resulted in remarkable reduction of the catalytic activity with E. coli and Arabidopsis thaliana ribonuclease Z (51, 52), human Artemis (53,54), Bacillus thuringiensis N-acyl-L-homoserine lactone hydrolase (55), and IMP-1 metallo-␤-lactamase (56 -58). Furthermore, the residue corresponding to Asp-147 (Artemis), that corresponding to His-190 (ribonuclease Z and IMP-1), and that corresponding to Asp-284 (ribonuclease Z, Artemis, and N-acyl-L-homoserine lactone hydrolase) were also considered to be catalytically important based on the results of mutagenesis (51)(52)(53)(54)(55)(56)(57)(58). On the other hand, contrary to our speculation, H331N still showed an activity higher than the other mutants (Table 6), suggesting the presence of another histidine residue as a substitute for His-331.…”

“…Furthermore, recent crystallographic studies revealed three-dimensional structures of members of the metallo-␤-lactamase family such as B. cereus ␤-lactamase (60), Fluoribacter gormanii Zn-␤-lactamase (61), human glyoxalase II (62), Desulfovibrio gigas rubredoxin oxygen:oxidoreductase (63), Bacillus subtilis and Thermotoga maritima ribonuclease Z (64,65), Streptococcus pneumoniae phosphorylcholine esterase (59), and B. thuringiensis N-acyl-L-homoserine lactone hydrolase (55). The results elucidated that the conserved domain within the family is composed of ␣␤/␤␣ sandwich structure.…”

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

“…AHLs are involved as signaling molecules in the suppression of quorum-sensing systems, 3,4 which are utilized by many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora. 5 All ZMH family members contain a conserved mononuclear or binuclear zinc binding site, characterized by the HxHxH motif.…”

The quorum-quenching N -acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket

“…A structurally well-characterized quorum-quenching enzyme is the N-acyl homoserine lactone lactonase of Bacillus thuringiensis (9,10), which belongs to the metallo-β-lactamase superfamily. This enzyme disrupts quorum sensing by cleaving the ester bond in the homoserine lactone ring, making it inactive in signaling.…”