PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal

Abstract

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.

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

“…PqsA converts anthranilic acid to anthraniloyl-CoA that is condensed with malonyl-CoA to form 2-aminobenzoylacetyl-CoA (2-ABA-CoA) in a reaction catalysed by PqsD [15,16]. The thioesterase activity of PqsE converts 2-ABA-CoA into 2-aminobenzoylacetate (2-ABA) [17]; HHQ is formed through the condensation of octanoyl-coenzyme A and 2-ABA via the PqsBC heterodimer [18,19]. …”

The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH‐like 2‐Heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) Biosynthesis Enzyme PqsBC

“…Owing to its instability, it can spontaneously decompose into 2‐aminoacetophenone (2‐AA) by the loss of CO 2 or by undergoing intramolecular cyclization into 2,4‐dihydroxyquinoline (DHQ) . In the main biosynthetic route to HHQ, however, the heterodimeric PqsBC complex (EC 2.3.1.180) transfers 2‐ABA onto octanoate to synthesize HHQ in a decarboxylative Claisen condensation after an acyl‐enzyme intermediate between the active‐site cysteine (C129) of PqsC and octanoyl‐CoA is formed . In a final step, HHQ is hydroxylated at the C3 position by the monooxygenase PqsH .…”

Structures of the N‐Terminal Domain of PqsA in Complex with Anthraniloyl‐ and 6‐Fluoroanthraniloyl‐AMP: Substrate Activation in Pseudomonas Quinolone Signal (PQS) Biosynthesis

“…Because the pqs system is unique to P. aeruginosa and has a central role in virulence, it might present an attractive target for new pathoblocker approaches. To this end, structural information provides important insight into the ligand‐binding properties of proteins, and since the discovery of the pqs system crystal structures of nearly all PQS biosynthetic proteins, with the exceptions of PqsA and PqsH, and of the ligand‐binding domain of the HHQ/PQS‐responsive transcription factor MvfR have become available . PqsH, however, seems to be an unpromising drug target, because it has been demonstrated that a P. aeruginosa PA14 pqsH mutant that is unable to transform HHQ into PQS exhibits virulence similar to that of the wild‐type strain .…”