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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

Structure and function of the GT domain.(a) Structure of the GT domain. Structure of Taka-amylase A (right, PDB 7TAA) is shown for reference. CSRI–IV in both structures are coloured in blue and cyan, respectively. Catalytic residues are highlighted. Subdomain B and the equivalent region in Taka-amylase A (domain B) are omitted for clarity. (b) Structure of the GT domain active-site pocket. The −1 subsite in Taka-amylase A (grey for the carbon atoms) is superimposed for reference. Amino acid residue labels on the second lines are for Taka-amylase A. Catalytic residues are labelled in red. The B-1 residue of the bound oligosaccharide B in the maltopentaose complex structure and the −1 saccharide unit of acarbose in the Taka-amylase A structure are shown. (c) GT activity of CgGDE and its mutants. The reactions catalysed by CgGDE and its mutants with maltopentaose as the substrate were analysed with thin-layer chromatography. In the control experiment (lane Ctrl) no CgGDE were added to the reaction. Oligosaccharides with 2–7 residues (G2–G7) were used as standards.
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f2: Structure and function of the GT domain.(a) Structure of the GT domain. Structure of Taka-amylase A (right, PDB 7TAA) is shown for reference. CSRI–IV in both structures are coloured in blue and cyan, respectively. Catalytic residues are highlighted. Subdomain B and the equivalent region in Taka-amylase A (domain B) are omitted for clarity. (b) Structure of the GT domain active-site pocket. The −1 subsite in Taka-amylase A (grey for the carbon atoms) is superimposed for reference. Amino acid residue labels on the second lines are for Taka-amylase A. Catalytic residues are labelled in red. The B-1 residue of the bound oligosaccharide B in the maltopentaose complex structure and the −1 saccharide unit of acarbose in the Taka-amylase A structure are shown. (c) GT activity of CgGDE and its mutants. The reactions catalysed by CgGDE and its mutants with maltopentaose as the substrate were analysed with thin-layer chromatography. In the control experiment (lane Ctrl) no CgGDE were added to the reaction. Oligosaccharides with 2–7 residues (G2–G7) were used as standards.

Mentions: Consistent with the modest sequence similarity between them, the structure of the CgGDE N-terminal region (residues 132–869) is homologous to that of GH13 family members, and CSRI–IV in them occupy similar locations (Fig. 2a). Like GH13 family members, this region can be further divided into three subdomains: a TIM barrel subdomain A followed by an all-β subdomain C, and a subdomain B inserted between β3 and α3 of subdomain A. Subdomains A and C are equivalent to domains A and C in GH13 family members. Subdomain B appears to adopt a novel fold, and the equivalent region in GH13 family members (domain B) is variable. Reactions catalysed by GH13 family members involve an initial cleavage of the glycosidic bond between the +1 and −1 residues of their polysaccharide substrates. They have a highly conserved catalytic core, the −1 subsite, which accommodates the −1 residue and provides the catalytic nucleophile and proton donor (for instance, Asp206 and Glu230 in Taka-amylase A)21. Despite the low overall sequence identities between CgGDE and GH13 family members (no more than 15%), the equivalent region in CgGDE adopts an almost identical structure, with Asp535 and Glu564 occupying equivalent locations as the catalytic nucleophile and proton donor, respectively (Fig. 2b).


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)

Structure and function of the GT domain.(a) Structure of the GT domain. Structure of Taka-amylase A (right, PDB 7TAA) is shown for reference. CSRI–IV in both structures are coloured in blue and cyan, respectively. Catalytic residues are highlighted. Subdomain B and the equivalent region in Taka-amylase A (domain B) are omitted for clarity. (b) Structure of the GT domain active-site pocket. The −1 subsite in Taka-amylase A (grey for the carbon atoms) is superimposed for reference. Amino acid residue labels on the second lines are for Taka-amylase A. Catalytic residues are labelled in red. The B-1 residue of the bound oligosaccharide B in the maltopentaose complex structure and the −1 saccharide unit of acarbose in the Taka-amylase A structure are shown. (c) GT activity of CgGDE and its mutants. The reactions catalysed by CgGDE and its mutants with maltopentaose as the substrate were analysed with thin-layer chromatography. In the control experiment (lane Ctrl) no CgGDE were added to the reaction. Oligosaccharides with 2–7 residues (G2–G7) were used as standards.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Structure and function of the GT domain.(a) Structure of the GT domain. Structure of Taka-amylase A (right, PDB 7TAA) is shown for reference. CSRI–IV in both structures are coloured in blue and cyan, respectively. Catalytic residues are highlighted. Subdomain B and the equivalent region in Taka-amylase A (domain B) are omitted for clarity. (b) Structure of the GT domain active-site pocket. The −1 subsite in Taka-amylase A (grey for the carbon atoms) is superimposed for reference. Amino acid residue labels on the second lines are for Taka-amylase A. Catalytic residues are labelled in red. The B-1 residue of the bound oligosaccharide B in the maltopentaose complex structure and the −1 saccharide unit of acarbose in the Taka-amylase A structure are shown. (c) GT activity of CgGDE and its mutants. The reactions catalysed by CgGDE and its mutants with maltopentaose as the substrate were analysed with thin-layer chromatography. In the control experiment (lane Ctrl) no CgGDE were added to the reaction. Oligosaccharides with 2–7 residues (G2–G7) were used as standards.
Mentions: Consistent with the modest sequence similarity between them, the structure of the CgGDE N-terminal region (residues 132–869) is homologous to that of GH13 family members, and CSRI–IV in them occupy similar locations (Fig. 2a). Like GH13 family members, this region can be further divided into three subdomains: a TIM barrel subdomain A followed by an all-β subdomain C, and a subdomain B inserted between β3 and α3 of subdomain A. Subdomains A and C are equivalent to domains A and C in GH13 family members. Subdomain B appears to adopt a novel fold, and the equivalent region in GH13 family members (domain B) is variable. Reactions catalysed by GH13 family members involve an initial cleavage of the glycosidic bond between the +1 and −1 residues of their polysaccharide substrates. They have a highly conserved catalytic core, the −1 subsite, which accommodates the −1 residue and provides the catalytic nucleophile and proton donor (for instance, Asp206 and Glu230 in Taka-amylase A)21. Despite the low overall sequence identities between CgGDE and GH13 family members (no more than 15%), the equivalent region in CgGDE adopts an almost identical structure, with Asp535 and Glu564 occupying equivalent locations as the catalytic nucleophile and proton donor, respectively (Fig. 2b).

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