Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Abstract

Yeast cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated ␤(1-3) glucan chains. However, the molecular mechanisms that control the balance between hydrolysis and transglycosylation in these enzymes are not understood. Here we present the first crystal structure of a glucan transglycosylase, Saccharomyces cerevisiae Gas2 (ScGas2), revealing a multidomain fold, with a (␤␣) 8 catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different ␤-glucan substrate/product oligosaccharides provide "snapshots" of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive ␤(1-3) glucan elongation. Together with mutagenesis and analysis of reaction products, the structures suggest a "base occlusion" mechanism through which these enzymes protect the covalent protein-enzyme intermediate from a water nucleophile, thus controlling the balance between hydrolysis and transglycosylation and driving the elongation of ␤(1-3) glucan chains in the yeast cell wall.The cell wall of fungal organisms is a dynamic structure, providing protection against hostile environments, yet also harboring many hydrolytic and toxic molecules required for the fungus to invade its ecological niche (1). Polysaccharides account for over 90% of the cell wall. The central skeletal component of the cell wall common to the vast majority of fungal species is a branched core of ␤(1,3) glucan, linked to chitin via a ␤(1,4) linkage (1). Interchain ␤(1,6) glucosidic linkages account for 3 and 4% of the total glucan linkages in Saccharomyces cerevisiae and Aspergillus fumigatus, respectively (2-4). This core is embedded in a complex of amorphous proteins and/or polysaccharide whose composition is highly species-dependent. The core ␤(1,3) glucan is subjected to continuous synthetic elaboration, degradation, and remodeling by a large arsenal of enzymes, whose activities must be appropriately balanced to provide the cell wall with adequate elasticity to allow growth, budding, or branching and yet sufficient strength to guard against cell lysis (1).Glucan synthase is a protein complex located at the plasma membrane, synthesizing ␤(1,3) glucan from UDP-glucose (65-90% of the total glucan). In cell wall remodeling, glycoside hydrolases and glycosyltransferases/transglycosylases play a crucial role (1, 5). Pure glycoside hydrolases degrade glycans mainly to regulate the plasticity of the cell wall under different circumstances, such as cell division, cell separation, and sporulation (5), whereas glycoside hydrolases with significant transglycosylase activity are capable of forming new glycosidic bonds between oligosaccharides, generating longer or branched polymers. Previous studies have shown...

Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p

“…While efficient glycosylation of Asn253 in Gas1p in vivo requires the CxxC motif of either Ost3p or Ost6p [23], it is not clear if this requirement is due to the formation of a mixed disulfide bond between Ost3p or Ost6p and either Cys74 or Cys421 in Gas1p, that our analyses here detected could bind in vitro. Both Cys74 and Cys421 are distant from Asn253 both in amino acid sequence and in the predicted final folded protein structure of Gas1p [37,38]. It is not clear if the peptides that we detected forming mixed disulfides with Ost3p and Ost6p in vitro also form such transient bonds in vivo, or if these particular bonds are key for glycosylation at Asn253 of Gas1p.…”

Sequence-based protein stabilization in the absence of glycosylation

“…Gas1p is a major cellwall protein during vegetative growth while its functionally equivalent paralogue Gas2p is predominantly expressed during sporulation 24,25 . Gas1p and Gas2p share 45% sequence identity, conserved secondary structure and identical disulphide bond connectivity 26,27 . However, Gas1p has 10 N-glycosylation sequons while Gas2p has only 1.…”

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

“…The Bgl2p, Crh1p, and Gas1p enzymes (glucanases/transglycosylases) have been shown to be involved in cell wall biogenesis by cross-linking the glucan-chitin matrix (37)(38)(39)(40)(41)(42)(43)(44)(45). These enzymes use a process similar to the one we propose for Dfg5p and Dcw1p to cross-link the glucan and chitin polymers (44,45).…”