The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.

中文翻译：

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纤维素 FRET-FLIM 和单分子追踪揭示了铜绿假单胞菌中 pyoverdine 生物合成酶的超分子结构

pyoverdine (PVD) 的生物合成铜绿假单胞菌涉及多个酶促步骤，包括非核糖体肽合成酶 (NRPS) 的作用。NRPS 的一个标志是它们能够利用由共表达的辅助酶合成的非蛋白氨基酸。一般认为次级代谢途径的不同酶组装成大的超分子复合物。然而，在细胞环境中组装顺序酶的证据很少。在这里，我们使用在蜂窝通过荧光寿命显微镜 (FRET-FLIM) 测量的单分子跟踪和 Förster 共振能量转移，以探索鸟氨酸羟化酶 PvdA 的空间分配及其与 NRPS 的相互作用。我们发现 PvdA 主要扩散到与细胞质中的大复合物结合，具有小的可交换捕获部分。FRET-FLIM 清楚地表明 PvdA 与 PvdJ、PvdI、PvdL 和 PvdD（参与 PVD ​​途径的四种 NRPS）物理相互作用铜绿假单胞菌PAO1。根据与之相互作用的 NRPS，PvdA 的结合模式明显不同，这表明 PvdA 结合位点与 NRPS 的酶活性位点共同进化。我们的数据为负责 PVD ​​生物合成的强组织多酶复合物提供了证据，并说明了结合位点如何进化以精细控制连续酶的共定位并促进代谢途径效率。