Abstract
A glucodextranase (iGDase) from Arthrobacter globiformis I42 hydrolyzes alpha-1,6-glucosidic linkages of dextran from the non-reducing end to produce beta-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-A resolution. The structure of iGDase is composed of four domains N, A, B, and C. Domain A forms an (alpha/alpha)(6)-barrel structure and domain N consists of 17 antiparallel beta-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.
Highlights
Glucodextranase (EC 3.2.1.70, GDase)1 hydrolyzes ␣-1,6-glucosidic linkages of dextran to release -D-glucose from the non-reducing end via an inverting reaction mechanism (1)
We found that the C-terminal portion of tGDase is weakly homologous with a surface layer homology (SLH) domain conserved in pullulanase from Thermococcus hydrothermalis EM1 (6) and amylopullulanase from Pyrococcus abyssi
In the classification of glycoside hydrolase (GH) family members based on the amino acid sequences (8 –10), GDase is classified into GH family 15, whose major members are GAs (EC 3.2.1.3)
Summary
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). Glucodextranase (EC 3.2.1.70, GDase) hydrolyzes ␣-1,6-glucosidic linkages of dextran to release -D-glucose from the non-reducing end via an inverting reaction mechanism (1). Arthrobacter globiformis I42, a Gram-positive bacterium, produces a glucodextranase (iGDase) as an exocellular enzyme (1) This enzyme hydrolyzes the ␣-1,4-glucosidic linkages of starch to produce -D-glucose, its activity for the ␣-1,4-glucosidic linkages is much less efficient than that for the ␣-1,6glucosidic linkages of dextran and isomaltooligosaccharides (2, 3). Dextranlyticum (12) have been reported as exo-type dextranases These exodextranases have, entirely different primary structures from iGDase and are categorized into GH families 27 and 49, respectively.
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