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 beta(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 (betaalpha)(8) catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different beta-glucan substrate/product oligosaccharides provide "snapshots" of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive beta(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 beta(1-3) glucan chains in the yeast cell wall.
Highlights
Embedded in a complex of amorphous proteins and/or polysaccharide whose composition is highly species-dependent
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
Product Binding as a “Base Occlusion” Mechanism to Protect against Hydrolysis—The Saccharomyces cerevisiae Gas2 (ScGas2)-laminaripentaose complex suggests that it is possible for both products of the initial step in hydrolysis/transglycosylation to remain associated with the enzyme, with an “occlusion” of the catalytic base by the product in the acceptor site, very similar to what has been observed for the PttXET16A-acceptor complex (Fig. 2)
Summary
Structures of ScGas 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, controlling the balance between hydrolysis and transglycosylation and driving the elongation of (1–3) glucan chains in the yeast cell wall. For laminarioligosaccharides with Ͼ10 sugars, these enzymes are able to cleave a (1–3) bond and transfer the newly formed reducing end (the “donor”) to the nonreducing end of another oligosaccharide (the “acceptor”) [6, 12, 13] This transferase reaction generates a new (1,3) linkage, resulting in the elongation of (1,3) glucan chains, offering a mechanism for the synthesis of longer glucan chains as alternative to, or in synergy with, glucan synthase. Site-directed mutagenesis data together with crystal structures of the ScGas2-oligosaccharide complexes shows that product binding in the acceptor site is crucial for tuning the balance between hydrolysis and transglycosylation
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