A novel fluorescence assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases

Abstract

The mechanical properties of fungal cell walls are largely determined by composition and mutual cross-linking of their macromolecular components. Previous work showed that the Crh proteins are required for the formation of cross-links between chitin and glucan at the Saccharomyces cerevisiae cell wall. In the present study, the proteins encoded by CRH1 and CRH2 were heterologously expressed in Pichia pastoris and a sensitive fluorescence in vitro soluble assay was devised for determination of their transglycosylating activities. Both proteins act as chitin transglycosylases; they use soluble chitin derivatives, such as carboxymethyl chitin, glycol-chitin and/or N-acetyl chito-oligosaccharides of DP (degree of polymerization)≥5 as the oligoglycosyl donors, and oligosaccharides derived from chitin, β-(1,3)-glucan (laminarin) and β-(1,6)-glucan (pustulan), fluorescently labelled with sulforhodamine or FITC as acceptors. The minimal number of intact hexopyranose units required by Crh1 and/or Crh2 in the molecule of the acceptor oligosaccharide was two and the effectivity of the acceptor increased with the increasing length of its oligosaccharide chain. Products of the transglycosylation reactions were hybrid molecules composed of the acceptor and portions of carboxymethyl chitin attached to its non-reducing end. Both proteins exhibited a weak chitinolytic activity in different assays whereby the ratio of endo- compared with exo-chitinase activity was approximately 4-fold higher in Crh1 than in Crh2. The pH optimum of both enzymes was 3.5 and the optimum temperature was 37°C. The results obtained in vitro with different fluorescently labelled oligosaccharides as artificial chitin acceptors corroborated well with those observed in vivo.