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Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases.

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Blanco, Noelia y Sanz, Ana Belén y Rodríguez Peña, José Manuel y Nombela, César y Farkaš, Vladimír y Hurtado Guerrero, Ramón y Arroyo, Javier (2015) Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases. The FEBS journal, 282 (4). pp. 715-31. ISSN 1742-464X

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URL Oficial: http://dx.doi.org/10.1111/febs.13176



Resumen

Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-β-d-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β-1,3-glucan, β-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.


Tipo de documento:Artículo
Palabras clave:Chitin, cell wall, carbohydrate binding module, computer modelling, glucan, glycoside hydrolase, transglycosylation
Materias:Ciencias Biomédicas > Farmacia > Microbiología
Código ID:33649
Depositado:26 Nov 2015 12:04
Última Modificación:26 Nov 2015 12:04

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