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 b-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-b-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 b-1,3-glucan, b-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.

The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall

“…Double deletion of GAS1 and CHS1 or CHS3 (involved in chitin synthesis) resulted in a lysed-bud or a severely compromised phenotype (44), suggesting that both branched β-(1,3)-glucan and chitin are important in the cell wall. However, no such lethality has been described for the CRH1 CRH2 double deletion (45) [Crh1p and Crh2p are involved in linking chitin to β-(1,3)-glucan and β-(1,6)-glucan (46, 47), respectively], suggesting that glucan-chitin linkage may not be an essential part of the cell wall fibrillar core.…”

‘Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity’

“…Therefore, Crh1 and Crh2 would act as transglycosylases operating via a double displacement mechanism with retention of the anomericity of the formed glycosidic bond (Davies and Henrissat, ; Rye and Withers, ; Sinnott, ). The transglycosylase reaction catalyzed by these proteins involves the cleavage of the β‐1,4 linkages of the chitin and subsequent attachment of the fragment of the donor molecule through the newly formed reducing end onto the OH‐group of the acceptor molecule, presumably by a β‐1,4‐glycosidic bond (Blanco et al ., ) (Fig. B).…”

“…Site‐directed mutagenesis of amino acid residues at the catalytic domain of S. cerevisiae CRH1 and CRH2 genes and a respective functional analysis revealed that conserved residues within the catalytic domain acting as nucleophile and general acid/base residues (Fig. B) are essential for their transglycosylase activity (Blanco et al ., ; Blanco et al ., ). Therefore, Crh1 and Crh2 would act as transglycosylases operating via a double displacement mechanism with retention of the anomericity of the formed glycosidic bond (Davies and Henrissat, ; Rye and Withers, ; Sinnott, ).…”

“…The nature of the newly formed bond is indicated by the fact that it can be hydrolyzed by purified chitinase, and it has been confirmed by MALDI‐TOF mass spectrometric analysis of the hybrid products (Mazán et al ., ). In contrast to other retaining glycosyl hydrolases that break down chitin, such as those of the GH18 family, the transglycosylase activity of the Crh proteins is not inhibited by allosamidin, excluding a substrate‐assisted catalytic mechanism (Blanco et al ., ). The conservation of the catalytic residues in the Crh orthologs of C. albicans , A. fumigatus , N. crassa and other fungi supports the assumption that transglycosylation is a common fungal mechanism for cross‐linking chitin to glucan through the action of these proteins.…”

“…A minimal size of three glucoses was required for the acceptor oligosaccharide and the cross‐linking efficiency increased with the length of the oligosaccharide chain from 3 to 7 (Cabib et al ., ). The cross‐linking of both glucans in this assay was dependent on both enzymes, Crh1 and Crh2, although to a higher extent on the latter, in agreement with a more relevant role of Crh2 in the cross‐linking (Blanco et al ., ; Mazán et al ., ). Moreover, the oligosaccharide incorporation was blocked in a crh1 Δ crh2 Δ strain.…”