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Publication Abstract from PubMed

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-beta-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the -1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.

Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis.,Collet L, Vander Wauven C, Oudjama Y, Galleni M, Dutoit R Acta Crystallogr D Struct Biol. 2021 Feb 1;77(Pt 2):205-216. doi:, 10.1107/S2059798320015557. Epub 2021 Jan 26. PMID:33559609^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.