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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

Substrate recognition by the GC domain active site.(a) Difference electron-density map for the oligosaccharide bound at the GC domain active site. (b) Accommodation of this oligosaccharide by the GC domain. (c) Specific debranching activities of CgGDE mutants R1123G and Y1407F, and their combinations with mutants possessing only the GT or GC activities.
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f5: Substrate recognition by the GC domain active site.(a) Difference electron-density map for the oligosaccharide bound at the GC domain active site. (b) Accommodation of this oligosaccharide by the GC domain. (c) Specific debranching activities of CgGDE mutants R1123G and Y1407F, and their combinations with mutants possessing only the GT or GC activities.

Mentions: At the GC domain active site an oligosaccharide with five residues can be fitted into the electron densities (Fig. 5a and Supplementary Fig. 5d). It binds into a cleft, with residues 2–3 interacting with Asn1114, Leu1115, Arg1123, Asn1125 and Asp1207, residues 4–5 interacting with His1066, Trp1075, Tyr1407, Tyr1424 and Asp1503, and the C6 hydroxyl of its residue 4 pointing towards the active-site pocket. This suggests that the cleft and the active-site pocket accommodate the GC substrate mainchain and its single-residue branch, respectively. This substrate accommodation presents the glycosidic bond to be cleaved (between the mainchain and the branch) to the GC active centre (Supplementary Fig. 5e). It indicates that the GC active site recognizes multiple residues in its substrate mainchain flanking the branch point, consistent with a study mapping the porcine GDE GC active-site structure29.


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)

Substrate recognition by the GC domain active site.(a) Difference electron-density map for the oligosaccharide bound at the GC domain active site. (b) Accommodation of this oligosaccharide by the GC domain. (c) Specific debranching activities of CgGDE mutants R1123G and Y1407F, 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

f5: Substrate recognition by the GC domain active site.(a) Difference electron-density map for the oligosaccharide bound at the GC domain active site. (b) Accommodation of this oligosaccharide by the GC domain. (c) Specific debranching activities of CgGDE mutants R1123G and Y1407F, and their combinations with mutants possessing only the GT or GC activities.
Mentions: At the GC domain active site an oligosaccharide with five residues can be fitted into the electron densities (Fig. 5a and Supplementary Fig. 5d). It binds into a cleft, with residues 2–3 interacting with Asn1114, Leu1115, Arg1123, Asn1125 and Asp1207, residues 4–5 interacting with His1066, Trp1075, Tyr1407, Tyr1424 and Asp1503, and the C6 hydroxyl of its residue 4 pointing towards the active-site pocket. This suggests that the cleft and the active-site pocket accommodate the GC substrate mainchain and its single-residue branch, respectively. This substrate accommodation presents the glycosidic bond to be cleaved (between the mainchain and the branch) to the GC active centre (Supplementary Fig. 5e). It indicates that the GC active site recognizes multiple residues in its substrate mainchain flanking the branch point, consistent with a study mapping the porcine GDE GC active-site structure29.

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