6ZZ3

RBcel1 cellulase variant Y201F with cellotriose covalently bound

Summary for 6ZZ3

• Entry DOI: 10.2210/pdb6zz3/pdb
• Related PRD ID: PRD_900014
• Descriptor: Endoglucanase, beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-alpha-D-glucopyranose, 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL, ... (4 entities in total)
• Functional Keywords: cellulase, complex, cellotriose, y201f variant, hydrolase
• Biological source: uncultured bacterium
• Total number of polymer chains: 4
• Total formula weight: 147087.65

Authors

Collet, L.,Dutoit, R. (deposition date: 2020-08-03, release date: 2021-07-21, Last modification date: 2024-01-31)

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 (2.095 Å)