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)-喹诺酮类(AQS)和2-烷基-4-羟基喹啉-N-氧化物(AQNO)作为信号分子参与细菌通讯(群体感应)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还不完全清楚。在我们先前项目的框架内，我们确定了PqsE是一种与先前的看法相反的酶，它参与了AQ的生物合成，并描述了它在调节分支AQ生物合成途径的不同产物水平中的作用。我们还研究了由辛酰辅酶A和2-氨基苯甲酰乙酸酯形成信号分子2-庚基-4(1H)-喹诺酮(HHQ)的缩合酶PqsBC的结构和反应机理，以及AQ途径的另一产物2-氨基苯乙酮对其竞争抑制作用。拟议项目的目的是确定形成主要最终产物所需的下游酶PqsH和PqsL的特征，分别是假单胞菌喹诺酮信号(PQS，2-庚基-3-羟基-4(1H)-喹诺酮)和呼吸抑制剂2-庚基-4-羟基喹啉-N-氧化物(HQNO)。单组分黄素单加氧酶PqsH催化HHQ羟化为PQS，可能与膜或膜蛋白有关。我们将确定野生型和截短型蛋白的亚细胞定位，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的活性是否受AQ途径的产物(如PqsBC所观察到的)的调节；在体内，这可能有助于平衡HHQ与PQS的水平。此外，我们还将分析同样被PqsH羟化的HQNO是否是PQS的另一个生理相关的前体。我们观察到，PqsL是合成AQNO所必需的黄素酶，需要黄素还原酶组分才能发挥活性，考虑到它属于UbiH家族，这是非常令人惊讶的。PqsL的底物仍不清楚，但PqsL反应似乎与PqsBC反应紧密耦合。PqsL的功能特征及其与PqsBC的相互作用，以及对PqsL与(共)底物复合体的结构的分析(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，并解决铜绿假单胞菌如何合成AQNO的长期存在的问题。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生物合成途径中的酶如何微调这些次生代谢产物的产生，将有助于我们进一步了解铜绿假单胞菌如何操纵其生物环境，并可能有助于抗毒力药物的开发。

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.