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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.

由机会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（AQNO）在细菌通讯（群体感应）中充当信号分子，或表现出抗微生物、免疫调节甚至多种活性。AQ生物合成途径的生物化学尚未完全了解。在我们先前项目的框架内，我们将PqsE鉴定为与先前的信念相反的酶，其作为途径特异性硫酯酶参与AQ生物合成，并且我们描述了其在调节分支AQ生物合成途径的不同产物的水平中的作用。我们还表征了缩合酶PqsBC的结构和反应机制，该酶从辛酰辅酶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），并描述了其被AQ途径的另一种产物2-氨基苯乙酮竞争性抑制。该项目旨在表征下游酶PqsH和PqsL，它们分别是形成主要终产物、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）所需的。单组分黄素单加氧酶PqsH催化HHQ羟基化为PQS，推测与膜或膜蛋白相关。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的活性是否受到AQ途径产物的调节（如对PqsBC所观察到的）;在体内，这可能有助于平衡HHQ与PQS的水平。此外，我们将分析是否HQNO，这也是羟基化的PqsH，是另一个生理相关的前体PQS。我们观察到，PqsL，一种AQNO合成所必需的黄素酶，需要黄素还原酶组分的活性，考虑到其与UbiH家族的联系，这是非常令人惊讶的。PqsL的底物仍然未知，然而，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PqsL与（共）底物复合物的结构分析（与Mattevi教授合作）将深入了解这种独特的黄素单加氧酶的作用方式，并解决长期存在的问题铜绿假单胞菌如何合成AQNO。PQS和HQNO显著促进铜绿假单胞菌的毒力和竞争力。因此，破译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推进我们对铜绿假单胞菌如何操纵其生物环境的认识，也可能有助于开发抗毒性剂。