Abstract
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.
Highlights
Carbohydrate-active enzymes (CAZy) are valuable alternative tools to the traditional chemical synthesis of glycans
We first chose the natural donor substrate Glc-1P and donor-like molecules, based on previous kinetics studies indicating that some modifications on the hexopyranose ring of the sugar 1-phosphate ligands have a significant impact on the enzymatic activity of cellodextrin phosphorylase (CDP).[11,16]
Our results indicate that for the molecular recognition of CDP acceptor regioisomers with a “d-glucose-β-(1-X)-d-glucose” sequence, CDP shows a preferential recognition for the α-anomer of those disaccharides with a β-(1-3) inter-glycosidic regiochemistry (d-laminaribiose), whereas this preference shifts toward the β-anomer when the inter-glycosidic regiochemistry is β-(1-4) (d-cellobiose)
Summary
Carbohydrate-active enzymes (CAZy) are valuable alternative tools to the traditional chemical synthesis of glycans. Structural details of the molecular recognition of donor and acceptor-like molecules with DP lower than 4 have yet to be reported, which are necessary to deepen our understanding of the molecular basis of such a broad specificity For weak binders, such as non-natural CDP ligands, structural information must be gained under the dynamic conditions existing in solution, which better reflect the rapid ligand binding kinetics which are not observable in the crystalline state employed for X-ray crystallography. We have previously used X-ray crystallography and saturation transfer difference NMR spectroscopy (STD NMR) to investigate substrate recognition with β-1,3 glucan phosphorylases.[12] Here, we have applied the high-resolution ligand-based NMR techniques STD NMR and transferred NOESY (tr-NOESY) experiments, in combination with molecular modelling calculations, to gain structural information on the interactions of CDP with donor- and acceptor-like ligands. Our study provides structural information at atomic detail that will inform the rational design of synthetic substrate analogues for CDP with appropriate decorations for the production of novel cellulosebased materials
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