Molecular Recognition of Natural and Non-Natural Substrates by Cellodextrin Phosphorylase from Ruminiclostridium Thermocellum Investigated by NMR Spectroscopy.
Molecular Recognition of Natural and Non-Natural Substrates by Cellodextrin Phosphorylase from Ruminiclostridium Thermocellum Investigated by NMR Spectroscopy.
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DOI：
10.1002/chem.202102039
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发表时间：
2021-11-11
影响因子：
4.3
通讯作者：
Angulo, Jesus
中科院分区：
文献类型：
--
作者：
Gabrielli, Valeria;Munoz-Garcia, Juan C.;Pergolizzi, Giulia;De Andrade, Peterson;Khimyak, Yaroslav Z.;Field, Robert A.;Angulo, Jesus
关键词：
β-1→4-葡聚糖多糖，如纤维素、衍生物和类似物，由于其独特的物理化学性质而引起人们的关注，作为生物技术中许多不同应用的理想候选物。大规模获得这些具有高纯度的多糖仍然具有挑战性，并且非常需要通过使用体外酶的生态友好替代品。 一种突出的候选酶是来自热纤瘤胃梭菌的纤维糊精磷酸化酶（CDP），其能够从短纤维糊精和α-d-葡萄糖1-磷酸（Glc-1-P）作为底物产生纤维素低聚物。值得注意的是，其对供体和受体的广泛特异性允许产生高度多样化的纤维素基结构，以生产新型材料。然而，为了充分利用这种CDP广泛的特异性，需要详细了解这种酶在溶液中对底物的分子识别。在此，我们通过饱和转移差（STD）NMR光谱，tr-NOESY和蛋白质-配体对接，详细研究了CDP在溶液中对配体的分子识别。我们的研究结果，在文献中以前的反应动力学数据的背景下进行了讨论，允许更好地理解这种生物技术相关酶的广泛结合特异性的结构基础。 了解热纤瘤胃梭菌纤维糊精磷酸化酶（CDP）广泛特异性的分子基础，可以促进高度多样化CDP催化的纤维素基结构的产生。在这里，NMR光谱和分子建模允许了解CDP如何能够将非天然化学修饰的供体和受体与不同的结合表位结合，揭示CDP配体的结构要求，这将使CDP能够开发生产具有高生物技术兴趣的新型材料。
β‐1→4‐Glucan polysaccharides like cellulose, derivatives and analogues, are attracting attention due to their unique physicochemical properties, as ideal candidates for many different applications in biotechnology. Access to these polysaccharides with a high level of purity at scale is still challenging, and eco‐friendly alternatives by using enzymes in vitro are highly desirable. One prominent candidate enzyme is cellodextrin phosphorylase (CDP) from Ruminiclostridium thermocellum, which is able to yield cellulose oligomers from short cellodextrins and α‐d‐glucose 1‐phosphate (Glc‐1‐P) as substrates. Remarkably, its broad specificity towards donors and acceptors allows the generation of highly diverse cellulose‐based structures to produce novel materials. However, to fully exploit this CDP broad specificity, a detailed understanding of the molecular recognition of substrates by this enzyme in solution is needed. Herein, we provide a detailed investigation of the molecular recognition of ligands by CDP in solution by saturation transfer difference (STD) NMR spectroscopy, tr‐NOESY and protein‐ligand docking. Our results, discussed in the context of previous reaction kinetics data in the literature, allow a better understanding of the structural basis of the broad binding specificity of this biotechnologically relevant enzyme. Understanding the molecular basis of the broad specificity of cellodextrin phosphorylase (CDP) from Ruminiclostridium thermocellum, can facilitate the generation of highly diverse CDP‐catalysed cellulose‐based structures. Here, NMR spectroscopy and molecular modelling allowed to understand how CDP is able to bind non‐natural chemically modified donors and acceptors with different binding epitopes, revealing the structural requirements for CDP ligands, which will enable exploitation of CDP to produce novel materials of high biotechnological interest.
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影响因子：
3.1
作者：
O'Neill EC;Pergolizzi G;Stevenson CEM;Lawson DM;Nepogodiev SA;Field RA
通讯作者：
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