Crystal Structure of the Covalent Intermediate of Amylosucrase from Neisseria polysaccharea
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- 作者：Malene H. Jensen ; Osman Mirza ; Cecile Albenne ; Magali Remaud-Simeon ; Pierre Monsan ; Michael Gajhede ; and Lars K. Skov
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 23, 2004
- 年：2004
- 卷：43
- 期：11
- 页码：3104 - 3110
- 全文大小：233K
- 年卷期：v.43,no.11(March 23, 2004)
- ISSN：1520-4995
文摘
The 
-retaining amylosucrase from the glycoside hydrolase family 13 performs a transferreaction of a glucosyl moiety from sucrose to an acceptor molecule. Amylosucrase has previously beenshown to be able to use -D-glucopyranosyl fluoride as a substrate, which suggested that it could also beused for trapping the reaction intermediate for crystallographic studies. In this paper, the crystal structureof the acid/base catalyst mutant, E328Q, with a covalently bound glucopyranosyl moiety is presented.Sucrose cocrystallized crystals were soaked with -D-glucopyranosyl fluoride, which resulted in the trappingof a covalent intermediate in the active site of the enzyme. The structure is refined to a resolution of 2.2Å and showed that binding of the covalent intermediate resulted in a backbone movement of 1 Å aroundthe location of the nucleophile, Asp286. This structure reveals the first covalent intermediate of an-retaining glycoside hydrolase where the glucosyl moiety is identical to the expected biologically relevantentity. Comparison to other enzymes with anticipated glucosylic covalent intermediates suggests that thisstructure is a representative model for such intermediates. Analysis of the active site shows howoligosaccharide binding disrupts the putative nucleophilic water binding site found in the hydrolases ofthe GH family 13. This reveals important parts of the structural background for the shift in function fromhydrolase to transglycosidase seen in amylosucrase.
-retaining amylosucrase from the glycoside hydrolase family 13 performs a transferreaction of a glucosyl moiety from sucrose to an acceptor molecule. Amylosucrase has previously beenshown to be able to use -D-glucopyranosyl fluoride as a substrate, which suggested that it could also beused for trapping the reaction intermediate for crystallographic studies. In this paper, the crystal structureof the acid/base catalyst mutant, E328Q, with a covalently bound glucopyranosyl moiety is presented.Sucrose cocrystallized crystals were soaked with -D-glucopyranosyl fluoride, which resulted in the trappingof a covalent intermediate in the active site of the enzyme. The structure is refined to a resolution of 2.2Å and showed that binding of the covalent intermediate resulted in a backbone movement of 1 Å aroundthe location of the nucleophile, Asp286. This structure reveals the first covalent intermediate of an-retaining glycoside hydrolase where the glucosyl moiety is identical to the expected biologically relevantentity. Comparison to other enzymes with anticipated glucosylic covalent intermediates suggests that thisstructure is a representative model for such intermediates. Analysis of the active site shows howoligosaccharide binding disrupts the putative nucleophilic water binding site found in the hydrolases ofthe GH family 13. This reveals important parts of the structural background for the shift in function fromhydrolase to transglycosidase seen in amylosucrase.