在纤维素中,FRET-FLIM和单分子跟踪揭示了铜绿假单胞菌中pyoverdine生物合成酶的超分子组织。

IF 7.2 2区 生物学 Q1 BIOPHYSICS Quarterly Reviews of Biophysics Pub Date : 2020-01-09 DOI:10.1017/S0033583519000155
Véronique Gasser, Morgane Malrieu, Anne Forster, Yves Mély, Isabelle J Schalk, Julien Godet
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引用次数: 7

摘要

铜绿假单胞菌生物合成吡啶(PVD)涉及多个酶促步骤,包括非核糖体肽合成酶(NRPSs)的作用。NRPS的一个特点是它们能够利用由共表达的辅助酶合成的非蛋白质原性氨基酸。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,在细胞环境中序列酶组装的证据很少。本文采用纤维素单分子跟踪和荧光寿命显微镜(FRET-FLIM)测量的Förster共振能量转移来探索鸟氨酸羟化酶PvdA的空间分布及其与NRPS的相互作用。我们发现PvdA主要是扩散结合到细胞质中的大复合物上,并有一小部分可交换捕获。FRET-FLIM清楚地显示PvdA与铜绿假单胞菌PAO1中参与PVD通路的四种NRPS PvdJ、PvdI、PvdL和PvdD存在物理相互作用。PvdA的结合模式因其与NRPS的相互作用而有显著差异,表明PvdA的结合位点与NRPS的酶活性位点共同进化。我们的数据为强组织的多酶复合物负责PVD的生物合成提供了证据,并说明了结合位点如何进化到精细地控制序列酶的共定位并促进代谢途径效率。
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In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa.

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.

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来源期刊
Quarterly Reviews of Biophysics
Quarterly Reviews of Biophysics 生物-生物物理
CiteScore
12.90
自引率
1.60%
发文量
16
期刊介绍: Quarterly Reviews of Biophysics covers the field of experimental and computational biophysics. Experimental biophysics span across different physics-based measurements such as optical microscopy, super-resolution imaging, electron microscopy, X-ray and neutron diffraction, spectroscopy, calorimetry, thermodynamics and their integrated uses. Computational biophysics includes theory, simulations, bioinformatics and system analysis. These biophysical methodologies are used to discover the structure, function and physiology of biological systems in varying complexities from cells, organelles, membranes, protein-nucleic acid complexes, molecular machines to molecules. The majority of reviews published are invited from authors who have made significant contributions to the field, who give critical, readable and sometimes controversial accounts of recent progress and problems in their specialty. The journal has long-standing, worldwide reputation, demonstrated by its high ranking in the ISI Science Citation Index, as a forum for general and specialized communication between biophysicists working in different areas. Thematic issues are occasionally published.
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