Structure Elucidation and Preliminary Assessment of Hydrolase Activity of PqsE, thePseudomonasQuinolone Signal (PQS) Response Protein

Abstract

In bacteria, the transcription of virulence genes is usually controlled by a cell density-dependent process known as "quorum sensing" (QS). QS relies on small diffusible signaling molecules that cross the bacterial cell wall and activate target transcription factors after a threshold concentration has been reached. Besides two hierarchical QS circuits based on N-acylhomoserine lactones, the human opportunistic pathogen Pseudomonas aeruginosa integrates a signaling system that depends on 2-heptyl-3-hydroxy-4-quinolone, termed "Pseudomonas quinolone signal" (PQS). PQS is produced from genes encoded in the pqs operon, which in addition to the biosynthetic enzymes PqsA-D contains a fifth gene, pqsE, that is not required for production of PQS but whose disruption leads to loss of signal transduction in several but not all pqs operon-dependent processes. PqsE was hence termed "PQS response protein", but its exact mechanism of action is unknown. We have determined the crystal structure of recombinant PqsE and show that it possesses a metallo-beta-lactamase fold with an Fe(II)Fe(III) center in the active site. A copurified ligand was assigned as benzoate and may indicate that PqsE exerts its regulatory effect by converting a chorismate-derived molecule. Further, PqsE was found to slowly hydrolyze phosphodiesters including single- and double-stranded DNA as well as mRNA and also the thioester S-(4-nitrobenzoyl)mercaptoethane. Higher activity was observed after incubation with Co(2+) and, to lesser entent, Mn(2+), suggesting that the Fe(II)Fe(III) center of recombinant PqsE may be an artifact of heterologous expression. A crystal complex of the E182A mutant with bis-pNPP was obtained and suggests a catalytic mechanism for hydrolysis.

Unravelling the Genome-Wide Contributions of Specific 2-Alkyl-4-Quinolones and PqsE to Quorum Sensing in Pseudomonas aeruginosa

“…aeruginosa transcriptome is unlikely to be direct, since this protein does not possess a DNA-binding domain [37]. Moreover, PqsE activity is not exclusively a consequence of its thioesterase activity since pqsE expression is sufficient to restore pyocyanin in an AQ-deficient ( pqsA mutant) background [6,11].…”

Regulation of Phenazine Biosynthesis

“…pqsE encodes a putative metallo-b-hydrolase of unknown function (Yu et al 2009) that appears to be ''caught'' in the positive feedback loop controlling HHQ production: it is induced as a result of this mechanism but is not required for HHQ synthesis. Nevertheless, PqsE is required for phenazine production in P. aeruginosa PAO1 and PA14 (Gallagher et al 2002;Recinos et al 2012).…”

“…Constitutive expression of PqsE in a pqsA mutant background is sufficient to promote phenazine production , suggesting that, in the context of phz operon expression, the positive feedback loop that promotes HHQ production serves the The mechanism whereby PqsE promotes phz operon expression remains completely undefined, as PqsE itself does not contain a DNA binding domain. PqsE may be involved in the transformation of an unknown signal (Yu et al 2009). We hypothesize that this signal controls the activity of an unidentified regulator of phz gene expression.…”

RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE

“…Transcription of pqsABCDE leads to the expression of the fifth gene in the operon, PqsE. The precise function of PqsE is not understood; however, structural analysis indicated that PqsE is related to the metallo-␤-lactamase superfamily and is not required for HAQ biosynthesis but independently regulates genes that are under PQS control (16)(17)(18). PqsE is required for swarming motility and biofilm formation and can activate virulence in the absence of a functional pqs system (19).…”