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

黄素单加氧酶 PqsH 和 PqsL 以及辅助蛋白，平衡铜绿假单胞菌产生的烷基羟基喹啉型群体感应信号和抗生素的水平

基本信息

批准号：
229433240
负责人：
Professorin Dr. Susanne Fetzner
金额：
--
依托单位：
Institut für Molekulare Mikrobiologie und Biotechnologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/229433240?language=en
关键词：
Flavin monooxygenases PqsH PqsL accessory

项目摘要

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.

2-烷基-4（1H） - 喹诺酮（AQS）和2-烷基-4-羟基喹啉-N-氧化物（AQNOS），由机会性病原体假单胞菌铜绿假单胞菌起到细菌通信中的信号分子（QUORUM传感）的信号分子（QUORUM传感）或具有抗微生物的抗微生物，或者是免疫元素的多种多样的，或者表现出抗微生物的模型。 AQ生物合成途径的生物化学尚不完全了解。在我们以前的项目的框架内，我们将PQSE确定为一种酶，与先前的信念相反，与先前的信念相反，与AQ生物合成为途径特异性硫酯酶，我们描述了其在调整分支AQ生物合成途径不同产物水平的作用。我们还表征了浓缩酶PQSBC的结构和反应机制，该酶PQSBC形成了来自Octanoyl-COA和2-氨基苯甲酰乙酸酯的信号分子2- heptyl-4（1H）-Quinolone（HHQ），并描述了其2-氨基烯酮的竞争性抑制作用，并描述了2-氨基烯酮的竞争性抑制作用。 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.单一组分黄素单加氧酶PQSH，催化HHQ至PQ的羟基化，大概与膜或膜蛋白有关。我们将确定野生型和截短蛋白的亚细胞定位，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的活性是否是通过AQ途径的产物调节的（如PQSBC所观察到的）；在体内，这可能有助于平衡HHQ与PQ的水平。此外，我们将分析PQSH也是羟基化的HQNO是否是PQS的另一个生理相关的前体。我们观察到PQSL是AQNO合成所需的黄酮酶，需要用于活性的黄素还原酶成分，考虑到其与UBIH家族的隶属关系，这是令人惊讶的。 PQSL的底物仍然未知，但是，PQSL反应似乎与PQSBC反应紧密耦合。 PQSL的功能表征，其与PQSBC的相互作用以及与（Co-）底物（与Mattevi教授合作）复杂的PQSL结构的分析，将洞悉这种独特的黄素单氧酶的作用方式，并解决了p. p. p. p. p. p. p. eruguginosa synthess nights nimate synthess symathes aqnos aqnos aqnos。 PQ和HQNO显着促进了铜绿假单胞菌的毒力和竞争力。因此，解密AQ生物合成途径的酶如何微调这些次级代谢产物的产生将提高我们对铜绿假单胞菌如何操纵其生物环境的知识，并可能有助于抗抗病剂的发展。