Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium capable of surviving in a broad range of natural environments and known to be involved in infectious diseases of various hosts. In humans, the opportunistic pathogen is one of the leading causes for nosocomial infections in immuno-compromised patients and is responsible for chronic lung infections in the majority of cystic fibrosis patients. The ability of P. aeruginosa to adapt to different environments and lifestyles is closely related to its ability to coordinate the survival strategy of a population by so-called quorum-sensing (QS) systems. QS is based on the production and release of small signaling molecules, called autoinducers, that increase in concentration as a function of cell density and activate corresponding transcriptional regulators after a threshold concentration has been reached. Three different QS systems are known from P. aeruginosa. The las and rhl systems use acyl-homoserine lactone (AHL) autoinducers and belong to the LuxI/LuxR-type systems that are widespread among Gram-negative bacteria. The third QS system is rather unique and restricted to particular Pseudomonas and Burkholderia strains. Therein 2-alkyl-4(1H) quinolones (AQ) autoinducers such as 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal: PQS) and its direct precursor 2-heptyl-4(1H)-quinolone (HHQ) are used (see Scheme 1). The pqs system is involved in the regulation of P. aeruginosa virulence such as pyocyanin biosynthesis, biofilm formation and maturation, the production of exoproducts like elastase, alkaline proteases, rhamnolipids, and hydrogen cyanide, and the expression of efflux pumps. Further, PQS itself can down-regulate the host-innate immune response. The sum of these effects makes the pqs system a highly attractive target for drug development to interfere with P. aeruginosa pathogenicity and biofilm formation. The general validity of this approach is supported by the results of several infection models in which PQS-deficient mutants show a reduced pathogenicity compared to P. aeruginosa wild type. A reduced pathogenicity was also observed in a mouse infection model when animals infected with the wild-type strain were treated with halogenated anthranilic acid derivatives that inhibit PQS biosynthesis. This treatment led to a significant increase in survival in comparison to the control group. To further explore this target it is necessary to understand the details of PQS formation in the pathogen. It is known that HHQ biosynthesis absolutely requires the genes pqsA–D, encoding an anthranilate:coenzyme A (CoA) ligase (pqsA) and three b-ketoacyl-acyl carrier protein synthase III (KAS III) homologues. An additional gene (pqsH), located apart from pqsA– D, is responsible for the hydroxylation of HHQ to form PQS. Feeding studies in vivo have demonstrated that HHQ most likely arises from "head-to-head" condensation of an anthraniloyl precursor and a b-keto fatty acid derivative (Scheme 1). However, the details of the enzymatic mechanism of this reaction and the nature of the b-keto fatty acid remained elusive. Furthermore, it has been shown in vitro that PqsA and PqsD catalyze the formation of 2,4-dihydroxyquinoline (DHQ), another secondary metabolite of P. aeruginosa. In DHQ biosynthesis PqsA activates anthranilic acid to anthraniloyl-CoA which is loaded to the active-site cysteine (C112) of PqsD, which itself catalyzes the decarboxylative Claisen condensation with malonyl-CoA. Based on the structural similarity between DHQ and HHQ, we reasoned that PqsD might be involved in a similar condensation reaction in HHQ biosynthesis. To prove this hypothesis, we heterologously expressed PqsD from strain PA14 in Escherichia coli and purified the enzyme for biochemical characterization in vitro. Anthraniloyl-CoA and three potential b-keto acid derivatives were chemically synthesized as substrates, including b-ketodecanoic acid (1), b-ketodecanoyl-CoA (2), and b-ketodecanoyl-N-acetylcysteamine thioester (3) as mimics of the hypothetical ACP-bound substrate (for details see the Supporting Information). The in vitro reaction contained recombinant PqsD, anthraniloyl-CoA, and one of the three b-keto acid substrates, and formation of the product HHQ was analyzed by high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS). HPLC-HRMS analysis confirmed HHQ formation in all three reactions by comparison of retention time, accurate mass, and fragmentation pattern to the synthetic reference compound (Figure 1), though high concentrations and longer incubation times were required for compounds 2 and 3 in comparison to the free acid. Thus, we provide direct experimental proof that PqsD catalyzes the condensation reaction between the anthraniloyl and the b-ketodecanoyl moieties in HHQ biosynthesis.