Flavin monooxygenases PqsH and PqsL and accessory proteins, balancing the levels of alkylhydroxyquinoline-type quorum sensing signals and antibiotics produced by Pseudomonas aeruginosa

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2-Alkyl-4(1H)-quinolones (AQs) and 2-alkyl-4-hydroxyquinoline-N-oxides (AQNOs) produced by the opportunistic pathogen Pseudomonas aeruginosa act as signal molecules in bacterial communication (quorum sensing) or exhibit antimicrobial, immune modulatory, or even multiple activities. The biochemistry of the AQ biosynthetic pathway is not fully understood. Within the frame of our previous project, we identified PqsE as an enzyme which contrary to the previous belief is involved in AQ biosynthesis as a pathway-specific thioesterase, and we described its role in tuning the levels of different products of the branched AQ biosynthetic pathway. We also characterized the structure and reaction mechanism of the condensing enzyme PqsBC, which forms the signal molecule 2-heptyl-4(1H)-quinolone (HHQ) from octanoyl-CoA and 2-aminobenzoylacetate, and described its competitive inhibition by 2-aminoacetophenone, another product of the AQ pathway. The proposed project aims at characterizing the downstream enzymes PqsH and PqsL which are required for formation of the major end products, the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone) and the respiratory inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), respectively. The single-component flavin monooxygenase PqsH, catalyzing the hydroxylation of HHQ to PQS, presumably is associated with the membrane or a membrane protein. We will determine the subcellular localization of wild-type and truncated protein and analyze whether factors interacting with PqsH affect its activity. We will also investigate whether the activity of PqsH is modulated by products of the AQ pathway (as observed for PqsBC); in vivo this could contribute to balancing the levels of HHQ vs. PQS. Furthermore, we will analyze whether HQNO, which is also hydroxylated by PqsH, is another physiologically relevant precursor of PQS. We observed that PqsL, a flavoenzyme necessary for AQNO synthesis, requires a flavin reductase component for activity, which considering its affiliation to the UbiH family is highly surprising. The substrate of PqsL still remains unknown, however, the PqsL reaction appears to be tightly coupled to the PqsBC reaction. Functional characterization of PqsL, its interaction with PqsBC, and analysis of the structure of PqsL in complex with (co-)substrates (cooperation with Prof. Mattevi) will provide insight into the mode of action of this unique flavin monooxygenase and solve the long-standing question of how P. aeruginosa synthesizes AQNOs. PQS and HQNO significantly contribute to the virulence and competitiveness of P. aeruginosa. Therefore, deciphering how the enzymes of the AQ biosynthetic pathway fine-tune the production of these secondary metabolites will advance our knowledge on how P. aeruginosa manipulates its biotic environment, and may also contribute to the development of anti-virulence agents.