Structural Insights into Substrate Specificity and Function of Glucodextranase

Abstract

A glucodextranase (iGDase) from Arthrobacter globiformis I42 hydrolyzes ␣-1,6-glucosidic linkages of dextran from the non-reducing end to produce ␤-D-glucose via an inverting reaction mechanism and classified into the glycoside hydrolase family 15 (GH15). Here we cloned the iGDase gene and determined the crystal structures of iGDase of the unliganded form and the complex with acarbose at 2.42-Å resolution. The structure of iGDase is composed of four domains N, A, B, and C. Domain A forms an (␣/␣) 6 -barrel structure and domain N consists of 17 antiparallel ␤-strands, and both domains are conserved in bacterial glucoamylases (GAs) and appear to be mainly concerned with catalytic activity. The structure of iGDase complexed with acarbose revealed that the positions and orientations of the residues at subsites ؊1 and ؉1 are nearly identical between iGDase and GA; however, the residues corresponding to subsite 3, which form the entrance of the substrate binding pocket, and the position of the open space and constriction of iGDase are different from those of GAs. On the other hand, domains B and C are not found in the bacterial GAs. The primary structure of domain C is homologous with a surface layer homology domain of pullulanases, and the three-dimensional structure of domain C resembles the carbohydrate-binding domain of some glycohydrolases.

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

“…The GH-15 thread coverage of ␤ is indicated in a two-color scheme, with blue and gray representing, respectively, the (␣/␣) 6 -barrel fold for the catalytic domain (A) and a mixed 2°structure corresponding to the B and C domains found in bacterial and archaeal glucoamy- lases and glucodextranases (61). The protein structurally closest to the best model of this region is Anthrobacter globiformus glucodextranase (PDB code 1ULV) (61), with a template modeling score of 0.8462 and root mean square deviation of 1.92 Å, indicating a good topographical match (62).…”

“…Both cryo-EM and SAXS analyses of the native (␣␤␥␦) 4 PhK complex show it to be a large Hierarchical protein structural modeling of the ␤ subunit was carried out using I-TASSER (46). X-ray crystal structures of Aspergillus awamori glucoamylase (blue and gray ribbon traces) and human PP2A PR65/A subunit (yellow trace) were used to thread, respectively, residues 41-670 and 671-1092 of the multidomain ␤ subunit primary sequence (61,65). The remaining N-terminal residues (1-40, blue trace) were modeled ab initio using QUARK (60).…”

Structural Similarity between a Starch-hydrolyzing Enzyme and an N-Glycan-Hydrolyzing Enzyme: Exohydrolases Cleaving α-1,X-Glucosidic Linkages to Produce β-Glucose

“…In contrast, glucodextranase is defined as an enzyme that hydrolyzes the α-1,6-glucosidic linkages of dextran from the non-reducing end to produce β-glucose. We have determined the enzymatic properties and the crystal structure of A. globiformis I42 glucodextranase (7,25), and indicated that this glucodextranase hydrolyzes the α-1,4-glucosidic linkages of maltotetraose and maltose; however, their activity of glucodextranase towards α-1,4-glucosidic linkage is about 1% of that for α-1,6-glucosidic linkage. The 3-dimensional structure of glucodextranase is composed of 4 domains, N, A, B, and C (Fig.…”

“…Most GH15 enzymes are identified as glucoamylases, but 2 glucodextranases (EC 3.2.1.70) from Arthrobacter globiformis strains (7,23), and one trehalase (EC 3.2.1.28) from Mycobacterium smegmatis (24) belong to GH15. While glucoamylases are specific for the hydrolysis of α-1,4-glucosidic linkages, they also cleave α-1,6-glucosidic linkage; however, their activity for the α-1,6-glucosidic linkage is 0.2% of that for the α-1,4-glucosidic linkage (14).…”

“…1A,B) (5,6), glucodextranase (EC 3.2.1.70; Fig. 1C) (7), and trehalase (EC 3.2.1.28; Fig. 1D) (8), which hydrolyzes α-1,4-, α-1,6-, and α-1,1-glucosidic linkages, respectively, and Escherichia coli YgjK (Fig.…”