Enzyme-Mediated Quenching of the Quinolone Signal (PQS): A Comparison between Naturally Occurring and Engineered PQS-Cleaving Dioxygenases.

The opportunistic pathogen employs quorum sensing to govern the production of many virulence factors. Interference with quorum sensing signaling has therefore been put forward as an attractive approach to disarm this pathogen. Here, we analyzed the quorum quenching properties of natural and engineered (2-alkyl-)3-hydroxy-4(1)-quinolone 2,4-dioxygenases (HQDs) that inactivate the signal molecule PQS ( quinolone signal; 2-heptyl-3-hydroxy-4(1)-quinolone).

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an α/β-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in of >2 °C, they all impaired catalytic activity.

An α/β-Hydrolase Fold Subfamily Comprising Quinolone Signal-Cleaving Dioxygenases.

The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/β-hydrolase fold superfamily. The α/β-hydrolase fold core domain consists of a β-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine.

Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the α/β-hydrolase fold family.

The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structures of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an α/β-hydrolase fold core domain with additional helices forming a cap domain.