Abstract
Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. Transglycosylases strengthen the cell wall by forming a rigid network of crosslinks through mechanisms that remain to be explored. Here we study the function of the Aspergillus fumigatus family of five Crh transglycosylases. Although crh genes are dispensable for cell viability, simultaneous deletion of all genes renders cells sensitive to cell wall interfering compounds. In vitro biochemical assays and localisation studies demonstrate that this family of enzymes functions redundantly as transglycosylases for both chitin-glucan and chitin-chitin cell wall crosslinks. To understand the molecular basis of this acceptor promiscuity, we solved the crystal structure of A. fumigatus Crh5 (AfCrh5) in complex with a chitooligosaccharide at the resolution of 2.8 Å, revealing an extensive elongated binding cleft for the donor (−4 to −1) substrate and a short acceptor (+1 to +2) binding site. Together with mutagenesis, the structure suggests a “hydrolysis product assisted” molecular mechanism favouring transglycosylation over hydrolysis.
Highlights
Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities
Sequence alignment of the A. fumigatus Crh enzymes with the S. cerevisiae and C. albicans
There have been few studies aimed at understanding cross-linking enzymes, limited to the Crh enzymes in S. cerevisiae and some other enzymes such as the large family of transglycosylases for elongating β-1,3-glucan (e.g. Gas family in S. cerevisiae[32], Gel family in A. fumigatus[33,34,35] and Phr family in C. albicans36), transglycosylases for branching β-1,3-glucan (e.g. Bgt family and Gel[4] in A. fumigatus32,37), and transglycosylases for covalent linking of galactomannan to the β-1,3-glucan–chitin cell wall core (e.g. Dfg family in A. fumigatus and N. crassa38)
Summary
Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. The demonstration that Crhs are required for cross-linking came from the analysis of isolated yeast cell walls, which were digested by β-1,3- or β-1,6-glucanases and subjected to size-exclusion chromatography after solubilisation by carboxymethylation to quantify the different glucan and chitin-associated fractions[5,6,7]. Using this approach it was shown that there is no chitin covalently bound to glucan in a strain deleted in CRH genes[6].
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