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Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations.

Zhai L, Feng L, Xia L, Yin H, Xiang S - Nat Commun (2016)

Bottom Line: These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition.The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity.Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

ABSTRACT
Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.

No MeSH data available.


Related in: MedlinePlus

Additional contacts of CgGDE with glycogen.(a–c) Structures of the additional oligosaccharide-binding sites in GT subdomain B (a), domains M2 (b) and GC (c). (d) Mutations at the additional oligosaccharide-binding sites decrease the affinity to glycogen. CgGDE pulled-down by glycogen immobilized on concanavalin A agarose was analysed by SDS PAGE. (e) Specific debranching activities of CgGDE mutants Y408A, W958A, D1400A and their combinations with mutants possessing only the GT or GC activities.
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f6: Additional contacts of CgGDE with glycogen.(a–c) Structures of the additional oligosaccharide-binding sites in GT subdomain B (a), domains M2 (b) and GC (c). (d) Mutations at the additional oligosaccharide-binding sites decrease the affinity to glycogen. CgGDE pulled-down by glycogen immobilized on concanavalin A agarose was analysed by SDS PAGE. (e) Specific debranching activities of CgGDE mutants Y408A, W958A, D1400A and their combinations with mutants possessing only the GT or GC activities.

Mentions: The maltopentaose complex structure revealed additional bound oligosaccharides. An oligosaccharide with four residues can be fitted into the electron densities at GT subdomain B (Supplementary Fig. 7a). Its residue 2 hydrogen bonds with the Asp412 side chain, residue 3 stacks against the Tyr408 side chain and forms additional interactions with Leu308 and Leu405, and residue 4 interacts with Asn401 (Fig. 6a). The electron densities at domain M2 are consistent with an oligosaccharide with four residues (Supplementary Fig. 7b). Residue 1 interacts with Ser913, residues 2 and 3 interact with the Tyr916 and Trp958 side chains, respectively. Hydrogen bonds are formed between residue 2 and the Asp917 mainchain carbonyl, and between residue 3 and the Asn952 side chain (Fig. 6b). An oligosaccharide with three residues can be fitted into the electron densities at domain GC ∼20 Å away from the substrate-binding site (Supplementary Fig. 7c). Residue 1 interacts with Tyr1351, residue 2 hydrogen bonds with the Asp1400 and Asn1402 side chains, residue 3 hydrogen bonds with the Ser1399 mainchain carbonyl and interacts with Asn1336 (Fig. 6c).


Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations.

Zhai L, Feng L, Xia L, Yin H, Xiang S - Nat Commun (2016)

Additional contacts of CgGDE with glycogen.(a–c) Structures of the additional oligosaccharide-binding sites in GT subdomain B (a), domains M2 (b) and GC (c). (d) Mutations at the additional oligosaccharide-binding sites decrease the affinity to glycogen. CgGDE pulled-down by glycogen immobilized on concanavalin A agarose was analysed by SDS PAGE. (e) Specific debranching activities of CgGDE mutants Y408A, W958A, D1400A and their combinations with mutants possessing only the GT or GC activities.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837477&req=5

f6: Additional contacts of CgGDE with glycogen.(a–c) Structures of the additional oligosaccharide-binding sites in GT subdomain B (a), domains M2 (b) and GC (c). (d) Mutations at the additional oligosaccharide-binding sites decrease the affinity to glycogen. CgGDE pulled-down by glycogen immobilized on concanavalin A agarose was analysed by SDS PAGE. (e) Specific debranching activities of CgGDE mutants Y408A, W958A, D1400A and their combinations with mutants possessing only the GT or GC activities.
Mentions: The maltopentaose complex structure revealed additional bound oligosaccharides. An oligosaccharide with four residues can be fitted into the electron densities at GT subdomain B (Supplementary Fig. 7a). Its residue 2 hydrogen bonds with the Asp412 side chain, residue 3 stacks against the Tyr408 side chain and forms additional interactions with Leu308 and Leu405, and residue 4 interacts with Asn401 (Fig. 6a). The electron densities at domain M2 are consistent with an oligosaccharide with four residues (Supplementary Fig. 7b). Residue 1 interacts with Ser913, residues 2 and 3 interact with the Tyr916 and Trp958 side chains, respectively. Hydrogen bonds are formed between residue 2 and the Asp917 mainchain carbonyl, and between residue 3 and the Asn952 side chain (Fig. 6b). An oligosaccharide with three residues can be fitted into the electron densities at domain GC ∼20 Å away from the substrate-binding site (Supplementary Fig. 7c). Residue 1 interacts with Tyr1351, residue 2 hydrogen bonds with the Asp1400 and Asn1402 side chains, residue 3 hydrogen bonds with the Ser1399 mainchain carbonyl and interacts with Asn1336 (Fig. 6c).

Bottom Line: These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition.The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity.Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

ABSTRACT
Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.

No MeSH data available.


Related in: MedlinePlus