Rhamnogalacturonan-I oligogalacturonide profiling, a tool to elucidate the mode of action of RGI-degrading enzymes and RGI structure

IF 10.7 1区化学 Q1 CHEMISTRY, APPLIED

Carbohydrate Polymers Pub Date : 2025-06-10 DOI:10.1016/j.carbpol.2025.123894

Adrien Lemaire , Catalina Duran-Garzon , Sébastien Rigaud , Pauline Trezel , Olivier Habrylo , Josip Safran , Aline Voxeur , Romain Roulard , Justine Miternique , Jean-Xavier Fontaine , Roland Molinié , Anaïs Guillaume , Loïc Levavasseur , Valérie Lefebvre , Serge Pilard , Jérôme Pelloux , Corinne Pau-Roblot

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引用次数: 0

Abstract

Pectins play a major role in the control of plant development and are widely used as hydrocolloids in the food industry. Yet, the fine structure of rhamnogalacturonan-I (RGI) pectic domain is difficult to determine owing to its chemical and structural complexity. In this study, we developed a sensitive analytical method based on the chromatographic separation of oligosaccharides derived from RGI, combined with accurate determination of their sizes and side-chains patterns using high resolution (HR) and tandem (MS/MS) mass spectrometry. This method revealed the structure of RGI from various sources following its hydrolysis by three enzymes, two rhamnogalacturonan hydrolase (RHG and RHG B) and one rhamnogalacturonan lyase (RGL) from A. Aculeatinus, that were heterologously expressed. We first defined the biochemical specificities of the recombinant RGI-ase, using enzymatic assays and NMR analyses, to ascertain their specific activities towards RGI. Finally, employing a specific library of >420 RGI-oligos, built from chromatography and mass spectrometry data, we assessed their mode of action by the analysis of the degradation products. Altogether, our results highlight that this method is of prime importance for analysis of RGI fine structure and to reveal differences in the enzyme's specificities related to peculiar structural motifs.

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鼠李糖半乳糖醛酸- i寡聚半乳糖醛酸分析，一种阐明RGI降解酶的作用模式和RGI结构的工具

果胶在控制植物发育方面起着重要作用，在食品工业中被广泛用作水胶体。然而，鼠李糖半乳糖醛酸- 1 （RGI）的精细结构由于其化学和结构的复杂性而难以确定。在这项研究中，我们建立了一种基于色谱分离的RGI低聚糖的灵敏分析方法，结合高分辨率（HR）和串联（MS/MS）质谱法精确测定其大小和侧链模式。本方法通过异源表达鼠李糖半乳糖酸水解酶（RHG和RHG B）和鼠李糖半乳糖酸裂解酶（RGL）三种酶的水解，揭示了不同来源RGI的结构。我们首先定义了重组RGI酶的生化特异性，使用酶分析和核磁共振分析，以确定它们对RGI的特异性活性。最后，利用从色谱和质谱数据建立的420 RGI-oligos特定文库，我们通过分析降解产物来评估它们的作用模式。总之，我们的结果突出表明，这种方法对于分析RGI精细结构和揭示与特殊结构基序相关的酶特异性差异至关重要。

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来源期刊

Carbohydrate Polymers 化学-高分子科学

CiteScore

22.40

自引率

8.00%

发文量

1286

审稿时长

47 days

期刊介绍： Carbohydrate Polymers stands as a prominent journal in the glycoscience field, dedicated to exploring and harnessing the potential of polysaccharides with applications spanning bioenergy, bioplastics, biomaterials, biorefining, chemistry, drug delivery, food, health, nanotechnology, packaging, paper, pharmaceuticals, medicine, oil recovery, textiles, tissue engineering, wood, and various aspects of glycoscience. The journal emphasizes the central role of well-characterized carbohydrate polymers, highlighting their significance as the primary focus rather than a peripheral topic. Each paper must prominently feature at least one named carbohydrate polymer, evident in both citation and title, with a commitment to innovative research that advances scientific knowledge.