Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene

Abstract

Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor p-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor /3-1,3 glucan, yielding a linear glucan containing a p-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429-4411, An apparent K,, of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent K, of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H,O competing for reaction at the activated donor site, thus resulting in hydrolysis.However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for p-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of p-1,3 glucan into the previously existing cell wall structure.Keywords. Glucosyltransferase ; fungal wall ; BGL2 protein ; wall assembly ; Saccharomyces.Synthesis and assembly of the fungal cell wall is a complex process about which we know much less compared to the analogous events occurring in bacteria. We do know that linear polymers of chitin, p-1,4 N-acetylglucosamine, and glucan, p-1,3 glucose, are synthesized by microsomal fractions prepared from fungal cells when iincubated in the presence of their appropriate UDP-sugars [l, 21. The process appears to be vectorial in that UDP-sugar presenf in the cytoplasm is polymerized, with concurrent translocation through the membrane of the growing polymeric chain. However, little if anything is known about the following aspects of p-1,3 polymer synthesis: (a) initiation of chain synthesis, (b) regulation of chain initiation, (c) translocation through the membrane, (d) mechanism and regulation of termination of chain synthesis, and (e) localization of chain synthesis during different phases of wall expansion. Furthermore, glucan found in the mature cell wall consists of highly branched molecules containing both p-1,3 and p-1,6 linkages.

Genome-wide Analysis of the Response to Cell Wall Mutations in the Yeast Saccharomyces cerevisiae

“…This is the case for PIR3, PST1, SED1, YGP1, and SPI1, also known to be induced under poor nutritional conditions (54,(73)(74)(75). Another very important subset of up-regulated genes are those encoding putative ␤-glucanosyl/glucosyltransferase activity (BGL2, CRH1, and SCW10 (76,77)) and ␤-1,3-glucanase (EXG1 (78)). This finding is in accordance with biochemical data showing that cell wall damage provokes an increase in the cross-linking between cell wall polymers and in particular between chitin and ␤-1,6-glucan (reviewed in Ref.…”

“…Note that, due to the gene similarity metric calculation used in the hierarchical clustering algorithm (39), a few sets of genes whose expression also changed in all mutants were spuriously dispersed all over the clustering representation. This was the case for BGL2, encoding a ␤-glucosyl transferase (77), and AFR1, encoding a protein involved in morphogenesis (79), as well as YBR230c, YHR138c, YMR191w, and YOR289w, which encode proteins with still undefined function. The second feature is related to the dendrograms on the horizontal axis, which depicts the relationships between cell wall mutations.…”

Differential regulation of cell wall biogenesis during growth and development in yeast

“…The major endoglucanase is encoded by BGL2. Bgl2p was later shown also to have transglucosylase activity, which is its predominant activity in the presence of relatively high concentrations of glucan non-reducing ends, as is the case in the cell wall (Goldman et al, 1995). In vitro, Bgl2p transfers 1,3-β-linked oligosaccharides of at least five residues to the non-reducing terminus of another 1,3-β-glucan chain, which is at least four residues long, and links them through a 1,6-β-linkage, thereby creating a kink.…”

Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae

“…Bgl2p is one of the most abundant cell-wall proteins (Mrša et al, 1993). It was characterized as an endo-1,3-b-glucanase and glucanosyltransferase that introduces b-1,6-linkages in 1,3-b-glucan chains (Mrša et al, 1993;Goldman et al, 1995). Deletion of BGL2 does not obviously affect cell morphology, whereas overexpression is harmful for cell viability (Mrša et al, 1993).…”