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436 a.a.
Key reference
DOI no: 10.1074/jbc.M807990200 J Biol Chem 284:8461-8469 (2009) PubMed id: 19097997
Molecular mechanisms of yeast cell wall glucan remodeling. R.Hurtado-Guerrero, A.W.Schüttelkopf, I.Mouyna, A.F.Ibrahim, S.Shepherd, T.Fontaine, J.P.Latgé, D.M.van Aalten. 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.
Selected figure(s)
Figure 2.
Structures of ScGas2-laminarioligosaccharide complexes. Stereo view of the active site of ScGas2 in complex with laminaripentaose and the hydrolysis products of laminariheptaose (i.e. laminaritetraose + laminaritriose), and comparison with PttXET16A bound to XLLG. The active site oriented to facilitate identification of the donor (left) and acceptor (right) subsites. The amino acids placed in the donor site and acceptor sites are shown as sticks with gray carbons. The residues targeted by site-directed mutagenesis, Gln^62, Tyr^107, Asp^132, Asn^175, Glu^176, Tyr^244, Glu^275, Tyr^307, Phe^404, and Tyr^474, are shown with orange carbon atoms. XLLG, laminaripentaose, laminaritetraose, and laminaritriose are represented as stick models with green carbon atoms. Protein-ligand and water-ligand hydrogen bonds are shown as dotted black lines. Water molecules involved in hydrogen bonds with the ligands are shown as cyan spheres. For clarity purposes, protein-water hydrogen bonds are not shown. Unbiased (i.e. before inclusion of any ligand model) |F[o]| – |F[c]|, ϕ[calc] electron density maps are shown at 2.5 σ.Figure 3.
High pressure liquid chromatography analysis of β(1,3) glucanosyltransferase/hydrolysis products. A, comparison of wild type ScGas2 kinetics against laminaripentaose and laminariheptaose, identifying laminaritetraose and laminaritriose as the main two degradation products of hydrolyzed laminariheptaose. B, product analysis from the incubation of the recombinant wild type ScGas2 and the following single mutant enzymes, Y107F, Y244Q, E275Q, and Y307Q, with 4 mm reduced G19 samples taken at the indicated time points.The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2009, 284, 8461-8469) copyright 2009. Figures were selected by an automated process.
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