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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 GC domain.(a) Structure of the GC domain. Structure of the Aspergillus awamori glucoamylase catalytic domain (right, PDB 1GAH) is shown for reference. Catalytic residues are highlighted. (b) Structure of the GC domain active-site pocket. Structure of the Aspergillus awamori glucoamylase is superimposed for reference (grey for the carbon atoms). Amino acid residue labels on the second lines are for glucoamylase. Catalytic residues are labelled in red. The +1 and −1 saccharide units of acarbose in the glucoamylase structure are shown. They mimic the +1 and −1 residues in its substrate, the glycosidic bond between which gets hydrolysed. (c) Specific debranching activities of CgGDE and its mutants. The specific activity is defined as the debranching reaction rate at the substrate concentration of 13 mg ml−1 (the reaction rate of the wild-type CgGDE plateaus at this substrate concentration, see Supplementary Fig. 4a), divided by the concentration of CgGDE. The error bars indicate standard deviations of triplicate experiments. The numbers above each bar indicate ratios to the wild-type value. ND indicates not detected. (d) Specific debranching activities of combinations of a GT-defective mutant and a GC-defective mutant. In reactions catalysed by a combination of mutants, they were added in a 1:1 ratio, and the concentration of one is used in calculating the specific activity. The activity of the wild-type CgGDE is shown for reference.
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f3: Structure and function of the GC domain.(a) Structure of the GC domain. Structure of the Aspergillus awamori glucoamylase catalytic domain (right, PDB 1GAH) is shown for reference. Catalytic residues are highlighted. (b) Structure of the GC domain active-site pocket. Structure of the Aspergillus awamori glucoamylase is superimposed for reference (grey for the carbon atoms). Amino acid residue labels on the second lines are for glucoamylase. Catalytic residues are labelled in red. The +1 and −1 saccharide units of acarbose in the glucoamylase structure are shown. They mimic the +1 and −1 residues in its substrate, the glycosidic bond between which gets hydrolysed. (c) Specific debranching activities of CgGDE and its mutants. The specific activity is defined as the debranching reaction rate at the substrate concentration of 13 mg ml−1 (the reaction rate of the wild-type CgGDE plateaus at this substrate concentration, see Supplementary Fig. 4a), divided by the concentration of CgGDE. The error bars indicate standard deviations of triplicate experiments. The numbers above each bar indicate ratios to the wild-type value. ND indicates not detected. (d) Specific debranching activities of combinations of a GT-defective mutant and a GC-defective mutant. In reactions catalysed by a combination of mutants, they were added in a 1:1 ratio, and the concentration of one is used in calculating the specific activity. The activity of the wild-type CgGDE is shown for reference.

Mentions: The C-terminal region of CgGDE (residues 1,023–1,528) adopts a (α/α)6-barrel structure, homologous to structures of the catalytic domain in glucoamylase (Fig. 3a) and other GH15 family members. These enzymes hydrolyse α-glycosidic bonds at the non-reducing end of polysaccharides, with two conserved acidic residues serving as the general acid and general base in the catalysis26. In Aspergillus awamori glucoamylase these (Glu179 and Glu400) and surrounding residues form a pocket accommodating the leaving glucosyl group27. Although the overall sequence identity between CgGDE and Aspergillus awamori glucoamylase is not significant (12%), the equivalent region in CgGDE forms an almost identical pocket, and Asp1241 and Glu1492 in it occupy equivalent locations as Aspergillus awamori glucoamylase residues Glu179 and Glu400 (Fig. 3b).


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 GC domain.(a) Structure of the GC domain. Structure of the Aspergillus awamori glucoamylase catalytic domain (right, PDB 1GAH) is shown for reference. Catalytic residues are highlighted. (b) Structure of the GC domain active-site pocket. Structure of the Aspergillus awamori glucoamylase is superimposed for reference (grey for the carbon atoms). Amino acid residue labels on the second lines are for glucoamylase. Catalytic residues are labelled in red. The +1 and −1 saccharide units of acarbose in the glucoamylase structure are shown. They mimic the +1 and −1 residues in its substrate, the glycosidic bond between which gets hydrolysed. (c) Specific debranching activities of CgGDE and its mutants. The specific activity is defined as the debranching reaction rate at the substrate concentration of 13 mg ml−1 (the reaction rate of the wild-type CgGDE plateaus at this substrate concentration, see Supplementary Fig. 4a), divided by the concentration of CgGDE. The error bars indicate standard deviations of triplicate experiments. The numbers above each bar indicate ratios to the wild-type value. ND indicates not detected. (d) Specific debranching activities of combinations of a GT-defective mutant and a GC-defective mutant. In reactions catalysed by a combination of mutants, they were added in a 1:1 ratio, and the concentration of one is used in calculating the specific activity. The activity of the wild-type CgGDE is shown for reference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4837477&req=5

f3: Structure and function of the GC domain.(a) Structure of the GC domain. Structure of the Aspergillus awamori glucoamylase catalytic domain (right, PDB 1GAH) is shown for reference. Catalytic residues are highlighted. (b) Structure of the GC domain active-site pocket. Structure of the Aspergillus awamori glucoamylase is superimposed for reference (grey for the carbon atoms). Amino acid residue labels on the second lines are for glucoamylase. Catalytic residues are labelled in red. The +1 and −1 saccharide units of acarbose in the glucoamylase structure are shown. They mimic the +1 and −1 residues in its substrate, the glycosidic bond between which gets hydrolysed. (c) Specific debranching activities of CgGDE and its mutants. The specific activity is defined as the debranching reaction rate at the substrate concentration of 13 mg ml−1 (the reaction rate of the wild-type CgGDE plateaus at this substrate concentration, see Supplementary Fig. 4a), divided by the concentration of CgGDE. The error bars indicate standard deviations of triplicate experiments. The numbers above each bar indicate ratios to the wild-type value. ND indicates not detected. (d) Specific debranching activities of combinations of a GT-defective mutant and a GC-defective mutant. In reactions catalysed by a combination of mutants, they were added in a 1:1 ratio, and the concentration of one is used in calculating the specific activity. The activity of the wild-type CgGDE is shown for reference.
Mentions: The C-terminal region of CgGDE (residues 1,023–1,528) adopts a (α/α)6-barrel structure, homologous to structures of the catalytic domain in glucoamylase (Fig. 3a) and other GH15 family members. These enzymes hydrolyse α-glycosidic bonds at the non-reducing end of polysaccharides, with two conserved acidic residues serving as the general acid and general base in the catalysis26. In Aspergillus awamori glucoamylase these (Glu179 and Glu400) and surrounding residues form a pocket accommodating the leaving glucosyl group27. Although the overall sequence identity between CgGDE and Aspergillus awamori glucoamylase is not significant (12%), the equivalent region in CgGDE forms an almost identical pocket, and Asp1241 and Glu1492 in it occupy equivalent locations as Aspergillus awamori glucoamylase residues Glu179 and Glu400 (Fig. 3b).

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