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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 GT domain active site.(a) Difference electron-density map for the oligosaccharides bound at the GT domain active site. The map shown here and in Fig. 5a was calculated before oligosaccharides were incorporated in the atomic model, and contoured at 2 σ. (b) Accommodation of oligosaccharide M by the GT domain active site. (c) Accommodation of oligosaccharide B by the GT domain active site. Part of the nearby oligosaccharide M is also shown. The +1 and −1 saccharide units of acarbose in the Taka-amylase A structure (PDB 7TAA, grey for the carbon atoms) is shown in partial transparency. They mimic the +1 and −1 residues in the substrate. (d) Specific debranching activities of the W470A mutant and its combinations with mutants possessing only the GT or the GC activities.
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f4: Substrate recognition by the GT domain active site.(a) Difference electron-density map for the oligosaccharides bound at the GT domain active site. The map shown here and in Fig. 5a was calculated before oligosaccharides were incorporated in the atomic model, and contoured at 2 σ. (b) Accommodation of oligosaccharide M by the GT domain active site. (c) Accommodation of oligosaccharide B by the GT domain active site. Part of the nearby oligosaccharide M is also shown. The +1 and −1 saccharide units of acarbose in the Taka-amylase A structure (PDB 7TAA, grey for the carbon atoms) is shown in partial transparency. They mimic the +1 and −1 residues in the substrate. (d) Specific debranching activities of the W470A mutant and its combinations with mutants possessing only the GT or the GC activities.

Mentions: The maltopentaose complex structure revealed a number of oligosaccharides bound at the GT and GC active sites, providing insights into their substrate recognition. At the GT active site two oligosaccharides, M and B (for mainchain and branch, respectively; see below), can be modelled into the electron densities, which contains 5 and 4 residues, respectively (Fig. 4a and Supplementary Fig. 5a). Oligosaccharide M interacts with subdomain B. Its concave face interacts with Trp470, Trp472 and Ile494, and its non-reducing end forms additional interactions with Gln421, Asn424, Arg425 and Tyr428 (Fig. 4b). Oligosaccharide B is buried deep into a cleft between subdomains A and B. Its B-1 residue is accommodated by the equivalent of the −1 subsite in GH13 members, but the positions of this residue and the −1 residue in the Taka-amylase A substrate28 are somewhat different (Fig. 2b). This is probably due to the lack of additional residues at its reducing end in the current structure. Residues B-2 to B-4 form extensive interactions with Glu188, Ser189, Ser191, Asn238, Pro450, Trp496, Asp498, Leu713 and Val714 (Fig. 4c). Structural comparison with Taka-amylase A structure28 indicates that a glucose residue can fit into the space between residues B-1 and M5 (Fig. 4c), suggesting the GT substrate mainchain and branch bind at where oligosaccharides M and B bind, respectively. Such substrate accommodation presents the glycosidic bond to be cleaved (between the last and second to last residues in the branch) to the GT active centre (Supplementary Fig. 5b). A cleft formed by residues Asn475, Pro476, Phe566 and Gly568 at the reducing end of oligosaccharide M might mediate additional interactions with the substrate mainchain (Fig. 4b).


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 GT domain active site.(a) Difference electron-density map for the oligosaccharides bound at the GT domain active site. The map shown here and in Fig. 5a was calculated before oligosaccharides were incorporated in the atomic model, and contoured at 2 σ. (b) Accommodation of oligosaccharide M by the GT domain active site. (c) Accommodation of oligosaccharide B by the GT domain active site. Part of the nearby oligosaccharide M is also shown. The +1 and −1 saccharide units of acarbose in the Taka-amylase A structure (PDB 7TAA, grey for the carbon atoms) is shown in partial transparency. They mimic the +1 and −1 residues in the substrate. (d) Specific debranching activities of the W470A mutant and its combinations with mutants possessing only the GT or the GC activities.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Substrate recognition by the GT domain active site.(a) Difference electron-density map for the oligosaccharides bound at the GT domain active site. The map shown here and in Fig. 5a was calculated before oligosaccharides were incorporated in the atomic model, and contoured at 2 σ. (b) Accommodation of oligosaccharide M by the GT domain active site. (c) Accommodation of oligosaccharide B by the GT domain active site. Part of the nearby oligosaccharide M is also shown. The +1 and −1 saccharide units of acarbose in the Taka-amylase A structure (PDB 7TAA, grey for the carbon atoms) is shown in partial transparency. They mimic the +1 and −1 residues in the substrate. (d) Specific debranching activities of the W470A mutant and its combinations with mutants possessing only the GT or the GC activities.
Mentions: The maltopentaose complex structure revealed a number of oligosaccharides bound at the GT and GC active sites, providing insights into their substrate recognition. At the GT active site two oligosaccharides, M and B (for mainchain and branch, respectively; see below), can be modelled into the electron densities, which contains 5 and 4 residues, respectively (Fig. 4a and Supplementary Fig. 5a). Oligosaccharide M interacts with subdomain B. Its concave face interacts with Trp470, Trp472 and Ile494, and its non-reducing end forms additional interactions with Gln421, Asn424, Arg425 and Tyr428 (Fig. 4b). Oligosaccharide B is buried deep into a cleft between subdomains A and B. Its B-1 residue is accommodated by the equivalent of the −1 subsite in GH13 members, but the positions of this residue and the −1 residue in the Taka-amylase A substrate28 are somewhat different (Fig. 2b). This is probably due to the lack of additional residues at its reducing end in the current structure. Residues B-2 to B-4 form extensive interactions with Glu188, Ser189, Ser191, Asn238, Pro450, Trp496, Asp498, Leu713 and Val714 (Fig. 4c). Structural comparison with Taka-amylase A structure28 indicates that a glucose residue can fit into the space between residues B-1 and M5 (Fig. 4c), suggesting the GT substrate mainchain and branch bind at where oligosaccharides M and B bind, respectively. Such substrate accommodation presents the glycosidic bond to be cleaved (between the last and second to last residues in the branch) to the GT active centre (Supplementary Fig. 5b). A cleft formed by residues Asn475, Pro476, Phe566 and Gly568 at the reducing end of oligosaccharide M might mediate additional interactions with the substrate mainchain (Fig. 4b).

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