Function and coordination of the periplasmic pyoverdine maturation proteins

吡咯烷酮是一种荧光铁载体，与致病假单胞菌的毒力有关，如铜绿假单胞菌或丁香假单胞菌。此外，有益的寄主相互作用，如荧光假单胞菌对植物生长的促进，得到了吡喃类化合物的支持，这有助于在铁有限的寄主环境中提供铁。在周质内由铁杆菌蛋白产生的吡喃啶类化合物，铁杆菌蛋白是细胞质中非核糖体合成肽。所有铁杆菌蛋白最初在其N-末端都有一个N-酰化的L-谷氨酸，然后是D-酪氨酸，L-2，4-二氨基丁酸酯，以及一个可变的、菌株特有的序列的进一步残基。在周质内，N-末端的谷氨酸经过脱酰化和进一步的化学修饰，由D-酪氨酸和L-2，4-二氨基丁酸酯生成二羟基喹啉荧光团，最终生成对Fe3+具有高亲和力的成熟吡喃。与荧光团的两个氧配体和多肽侧链的功能分泌的吡喃啶螯合物Fe3+。铁络合物被吸收到周质中，铁被还原释放并运输到细胞质中，游离的吡喃啶被回收利用。PvdQ(脱酰化)、PvdP(荧光团形成)、PvdO(荧光团形成)、PvdN(谷氨酸脱羧基和氧化)、PtaA(谷氨酸转氨酶)和PvdM(为PvdP提供铁蛋白)参与了周质成熟过程。在这个项目开始之前，只知道PvdP和PvdQ的功能。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。我们还可以更准确地确定pvdP的功能，检测pvdQ和pvdM突变体，并首次对pvdM进行生化分析。我们选择了基因组测序、遗传和制备良好的S1生物荧光假单胞菌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吡哆碱成熟途径。在过去的几年里，我们已经为所有编码吡喃类成熟蛋白的基因产生了框架缺失突变，并建立了功能互补系统，开发了表达系统，建立了吡喃类化合物和生物发生中间体的纯化和鉴定的基本方法。通过这个项目，我们现在将回答更多关于周质体内吡啶类化合物成熟的关键问题：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。相互作用研究将表明，底物移交或通道机制是否可以防止中间体的过早损失。因此，我们非常有信心通过这个项目来阐明周质内吡喃甲醚成熟过程的迷人和数十年的神秘过程。

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.