Function and coordination of the periplasmic pyoverdine maturation proteins

周质pyoverdine成熟蛋白的功能和协调

• 批准号: 387581586
• 负责人: Professor Dr. Thomas Brüser
• 金额: --
• 依托单位: Institut für Mikrobiologie (ifmb)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/387581586?language=en
• 关键词: Function; coordination; periplasmic; pyoverdine; maturation

Pyoverdines are fluorescent siderophores with fundamental relevance for the virulence of pathogenic pseudomonads, such as P. aeruginosa or P. syringae. Also, beneficial host interactions, such as the plant growth promotion by P. fluorescens, are supported by pyoverdines, which contribute to iron supply in iron-limited host environments. Pyoverdines are generated inside the periplasm from ferribactins, which are cytoplasmically non-ribosomally synthesized peptides. All ferribactins initially possess an N-acylated L-glutamate at their N-terminus, followed by a D-tyrosine, L-2,4-diaminobutyrate, and further residues of a variable, strain-specific sequence. Inside the periplasm, the N-terminal glutamate is deacylated and chemically further modified, and the dihydroxyquinoline fluorophore is generated from D-tyrosine and L-2,4-diaminobutyrate, which ultimately results in mature pyoverdine with its high affinity for Fe3+. Secreted pyoverdine chelates Fe3+ with the two oxygen ligands of the fluorophore and functions of the peptide side chains. The iron-complex is taken up into the periplasm, the iron is reductively released and transported into the cytoplasm, and the free pyoverdine is recycled. Six proteins are involved in the periplasmic maturation process: PvdQ (deacylation), PvdP (fluorophore-formation), PvdO (fluorophore-formation), PvdN (decarboxylation and oxygenation of the glutamate), PtaA (transamination of the glutamate) und PvdM (Ferribactin supply for PvdP). Before this project started, only the functions of PvdP and PvdQ were known. In the course of this project, we could reveal the functions of PtaA, PvdO, and PvdM, with PtaA being newly discovered by us. We could also more precisely define the function of PvdP, examine pvdQ and pvdM mutants, and we carried out first biochemical analyses of PvdM. We chose the genome sequenced, genetically and preparatively good amenable S1 organism P. fluorescens A506 as model system, knowing that other fluorescent pseudomonads in principle use the same pyoverdine maturation pathway. In the last years, we have generated in frame deletion mutants for all genes encoding the pyoverdine maturation proteins, and we have generated functional complementation systems, developed expression systems and established the basic methods for the purification and identification of pyoverdines and biogenesis intermediates. With this project, we will now answer more key questions of the periplasmic pyoverdine maturation: We will elucidate the function of PvdQ in more detail, clarify the copper assembly process for PvdP, analyze the redox activity of PvdO, characterize PtaA, and clarify the mechanism of PvdM function. Interaction studies will show, whether substrate handover or channeling mechanisms can prevent the premature loss of intermediates. We are thus very confident to clarify with this project the fascinating and over decades enigmatic process of periplasmic pyoverdine maturation.

绿脓菌荧光素是荧光铁载体，与致病性假单胞菌（如铜绿假单胞菌或铜绿假单胞菌）的毒力具有根本相关性。此外，有益的宿主相互作用，如荧光假单胞菌对植物生长的促进作用，得到了绿脓菌荧光素的支持，这有助于在铁有限的宿主环境中提供铁。绿脓菌荧光素在周质内由铁细菌素产生，铁细菌素是细胞质非核糖体合成的肽。所有铁杆菌素最初在其N-末端具有N-酰化L-谷氨酸，随后是D-酪氨酸、L-2，4-二氨基丁酸和可变的菌株特异性序列的其他残基。在周质内部，N-末端谷氨酸被脱酰化并进一步化学修饰，并且二羟基喹啉荧光团由D-酪氨酸和L-2，4-二氨基丁酸产生，这最终导致成熟的绿脓菌荧光素，其对Fe 3+具有高亲和力。分泌的绿脓菌荧光素与荧光团的两个氧配体和肽侧链的功能螯合Fe 3+。铁复合物被吸收到周质中，铁被还原释放并转运到细胞质中，游离的绿脓菌荧光素被回收。六种蛋白质参与周质成熟过程：PvdQ（脱酰作用）、PvdP（荧光团形成）、PvdO（荧光团形成）、PvdN（谷氨酸的脱羧和氧化）、PtaA（谷氨酸的转氨作用）和PvdM（PvdP的铁菌素供应）。在这个项目开始之前，只有PvdP和PvdQ的功能是已知的。在这个项目的过程中，我们可以揭示PtaA，PvdO和PvdM的功能，其中PtaA是我们新发现的。我们还可以更精确地定义PvdP的功能，检查pvdQ和pvdM突变体，并且我们对PvdM进行了第一次生化分析。我们选择基因组测序的、遗传上和免疫学上良好顺从的S1生物体荧光假单胞菌A506作为模型系统，知道其他荧光假单胞菌原则上使用相同的绿脓菌荧光素成熟途径。在过去的几年中，我们已经产生了编码绿脓菌荧光素成熟蛋白的所有基因的框内缺失突变体，并且我们已经产生了功能互补系统，开发了表达系统，并建立了用于纯化和鉴定绿脓菌荧光素和生物合成中间体的基本方法。通过这个项目，我们现在将回答周质绿脓菌荧光素成熟的更多关键问题：我们将更详细地阐明PvdQ的功能，阐明PvdP的铜组装过程，分析PvdO的氧化还原活性，表征PtaA，并阐明PvdM功能的机制。相互作用的研究将表明，无论是基板移交或沟道机制可以防止过早损失的中间体。因此，我们非常有信心通过这个项目来阐明周质绿脓菌荧光素成熟的迷人和数十年的神秘过程。