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

条件致病菌铜绿假单胞菌产生的2-烷基-4(1H)-喹诺酮（AQs）和2-烷基-4-羟基喹啉- n -氧化物（AQNOs）作为细菌交流（群体感应）的信号分子或具有抗菌、免疫调节甚至多重活性。AQ生物合成途径的生物化学机制尚不完全清楚。在我们之前的项目框架内，我们发现PqsE是一种酶，与之前的观点相反，它是一种途径特异性硫酯酶，参与AQ生物合成，我们描述了它在调节分支AQ生物合成途径不同产物水平方面的作用。我们还表征了由辛烷酰辅酶a和2-氨基苯甲酰乙酸形成信号分子2-庚基-4(1H)-喹诺酮（HHQ）的凝聚酶PqsBC的结构和反应机理，并描述了其与AQ途径的另一产物2-氨基苯乙酮的竞争性抑制作用。该项目旨在表征主要终产物喹诺酮假单胞菌信号（PQS， 2-heptyl-3-羟基-4(1H)-喹诺酮）和呼吸抑制剂2-heptyl-4-羟基喹啉- n -氧化物（HQNO）形成所需的下游酶PqsH和PqsL。单组分黄素单加氧酶PqsH，催化HHQ羟基化成PQS，推测与膜或膜蛋白有关。我们将确定野生型和截断蛋白的亚细胞定位，并分析与PqsH相互作用的因素是否影响其活性。我们还将研究PqsH的活性是否被AQ途径的产物调节（正如在PqsBC中观察到的那样）；在体内，这可能有助于平衡HHQ和PQS的水平。此外，我们将分析同样被PqsH羟基化的HQNO是否是PQS的另一个生理相关前体。我们观察到PqsL是一种合成AQNO所必需的黄酮酶，它需要黄素还原酶成分才能发挥活性，考虑到它与UbiH家族的关系，这是非常令人惊讶的。PqsL的底物尚不清楚，但PqsL反应似乎与PqsBC反应紧密耦合。PqsL的功能表征及其与PqsBC的相互作用，以及PqsL与（共）底物复合物的结构分析（与Mattevi教授合作）将有助于深入了解这种独特的黄素单加氧酶的作用模式，并解决P. aeruginosa如何合成AQNOs的长期问题。PQS和HQNO对铜绿假单胞菌的毒力和竞争力有显著影响。因此，破译AQ生物合成途径的酶如何微调这些次生代谢物的产生将有助于我们了解铜绿假单胞菌如何操纵其生物环境，并可能有助于抗毒剂的开发。

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.