A Novel β-( ,  ,  )-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus

Hartland, Robbert P.; Fontaine, Thierry; Debeaupuis, J P; Simenel, Catherine; Delepierre, Muriel; Latgé, Jean‐Paul

doi:10.1074/jbc.271.43.26843

Cited by 113 publications

(115 citation statements)

References 39 publications

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“…The HPAEC profile of rG13 at t 0 is shown in the upper left part of Figure 4. Gas1 produced smaller and larger oligosaccharides that were detected during the first 1-4 h of incubation and corresponded to the released and transferred products, as previously characterized (Hartland et al, 1996). The major initial products were rG7, rG8, rG18 and r G19 and confirmed the two-step enzyme activity:…”

Section: Enzymatic Activity Of Recombinant Proteinssupporting

confidence: 79%

“…Then, β-(1,3)-glucan chains are branched, elongated and remodelled in order to create a robust texture capable of counteracting the high internal pressure and determining cell morphology (Lesage and Bussey, 2006). A few years ago, a β-(1,3)-glucan elongase activity was first identified in Aspergillus fumigatus and then in Saccharomyces cerevisiae and Candida albicans (Hartland et al, 1996;Mouyna et al, 2000aMouyna et al, , 2005. The enzyme from A. fumigatus was named glucan-elongating protein 1 (Gel1).…”

Section: Introductionmentioning

confidence: 99%

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The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis

Ragni

Fontaine

Gissi

et al. 2007

Yeast

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108

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The GAS multigene family of Saccharomyces cerevisiae is constituted by five genes (GAS1-GAS5 ), but GAS1 was the only one to have been characterized to date. Gas1 is a glycosylphosphatidylinositol-anchored protein predominantly localized in the plasma membrane and is also a representative of family GH72 of glycosidase/transglycosidases, a wide group of yeast and fungal enzymes involved in cell wall assembly. Gas1-Gas5 proteins share a common N-terminal domain but exhibit different C-terminal extensions, in which a domain named Cys-Box is located. This domain is similar to the carbohydrate binding module 43 and is present only in Gas1p and Gas2p. Here we report the expression in P. pastoris of soluble forms of Gas proteins. Gas1, 2, 4 and 5 proteins were secreted with a yield of about 30-40 mg/l of medium, whereas the yield for Gas3p was about three times lower. Gas proteins proved to be N-glycosylated. Purified Gas proteins were tested for enzymatic activity. Gas2, Gas4 and Gas5p showed a β-(1,3)-glucanosyltransferase activity similar to Gas1p. A phylogenetic tree of the N-terminal regions of family GH72 members was constructed. Two subfamilies of N-terminal regions were distinguished: one subfamily, GH72 + , contains proteins that possess a Cys-box in the C-terminal region, whereas family GH72 − comprises proteins that lack a Cys-box. On the basis of this net distinction, we speculate that the type of C-tail region imposed constraints to the evolution of the N-terminal portion.

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Section: Enzymatic Activity Of Recombinant Proteinssupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis

Ragni

Fontaine

Gissi

et al. 2007

Yeast

Self Cite

108

139

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“…GH families can be classified as inverting or retaining enzymes based on the distance between the two catalytic residues, with inverting enzymes giving a distance of 10 Ϯ 2 Å on average, although retaining enzymes have the two residues located ϳ5.5 Å apart (45). In the ScGas2 crystal structure, Glu 176 and Glu 275 are 5.1 Å apart, suggesting the active site structure is compatible with a retaining mechanism, in agreement with previously published product analysis of ScGas2 (21). Given the sequence conservation of these residues, this will extend to all GH72 members.…”

Section: Cbm43 Domain Contains a Conserved Cysteine Structure Andsupporting

confidence: 73%

“…Hydrolysis experiments show that laminaripentaose is not hydrolyzed by ScGas2 (Fig. 3A), although a previous study of the A. fumigatus Gel1 ScGas2 orthologue showed that laminaripentaose was the smallest laminarioligosaccharide acceptor used by the enzyme (21). Surprisingly, soaking experiments with ScGas2 revealed not only an ordered laminaripentaose bound to the acceptor site, but also a (nonconvalently bound) laminaripentaose bound in the donor site (Fig.…”

Section: Cbm43 Domain Contains a Conserved Cysteine Structure Andmentioning

confidence: 84%

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Hurtado‐Guerrero

Schüttelkopf

Mouyna

et al. 2009

Journal of Biological Chemistry

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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...

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“…However, some fungal genes are involved in the remodeling of ␤-(1,3)-glucan were not identified in any Malassezia genome. Particularly, Gas/Gel proteins are ␤-(1,3)-glucanosyltransferases that are involved in the elongation of ␤-(1,3)-glucan chains (50,51). Sun proteins have been recently described to bind and degrade cell wall ␤-(1,3)-glucan (52).…”

Section: Discussionmentioning

confidence: 99%

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

Stalhberger

Simenel

Clavaud

et al. 2014

Journal of Biological Chemistry

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Background: Cell wall of Malassezia restricta is involved in interactions with human skin. Results: Its core is composed of cross-linked polysaccharides such as chitin, chitosan, ␤-(1,3)-glucan and ␤-(1,6)-glucan. Conclusion: The composition of cell wall polysaccharides of M. restricta is unique in the fungal kingdom. Significance: The cell wall of M. restricta has evolved as a yeast that adapted to the skin microenvironment and host interactions.

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A Novel β-( , , )-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus

Cited by 113 publications

References 39 publications

The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis

The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

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