title: Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases.
abstract: 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.
authors.affiliation: Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, IRYCIS, Spain.
authors.forename: Noelia
authors.initials: N
authors.lastname: Blanco
authors.forename: Ana B
authors.initials: AB
authors.lastname: Sanz
authors.forename: Jose M
authors.initials: JM
authors.lastname: Rodríguez-Peña
authors.forename: César
authors.initials: C
authors.lastname: Nombela
authors.forename: Vladimír
authors.initials: V
authors.lastname: Farkaš
authors.forename: Ramón
authors.initials: R
authors.lastname: Hurtado-Guerrero
authors.forename: Javier
authors.initials: J
authors.lastname: Arroyo
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D002686:Chitin / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D002686:Chitin; C407735:CRH1 protein, S cerevisiae; D006026:Glycoside Hydrolases; C407734:UTR2 protein, S cerevisiae
keywords: carbohydrate binding module; cell wall; chitin; computer modelling; glucan; glycoside hydrolase; transglycosylation