5LJF

Crystal structure of the endo-1,4-glucanase RBcel1 E135A with cellotriose

Summary for 5LJF

• Entry DOI: 10.2210/pdb5ljf/pdb
• Related: 4EE9 4M24
• Related PRD ID: PRD_900021
• Descriptor: Endoglucanase, beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose (3 entities in total)
• Functional Keywords: glycosyl hydrolase family 5, cellulase, tim barrel, beta-1, 4-endoglucanase, hydrolase
• Biological source: uncultured bacterium
• Total number of polymer chains: 2
• Total formula weight: 73656.91

Authors

Dutoit, R.,Collet, L.,Galleni, M.,Bauvois, C. (deposition date: 2016-07-18, release date: 2017-08-02, Last modification date: 2024-10-16)

Primary citation

Collet, L.,Vander Wauven, C.,Oudjama, Y.,Galleni, M.,Dutoit, R.
Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis.
Acta Crystallogr D Struct Biol, 77:205-216, 2021

PubMed Abstract: 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-β-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.

PubMed: 33559609
DOI: 10.1107/S2059798320015557

Experimental method

X-RAY DIFFRACTION (1.73439601224 Å)