Glycogen debranching enzyme (GDE) in mammals and yeast exhibits alpha-1,4-transferase and alpha-1,6-glucosidase activities within a single polypeptide chain and facilitates the breakdown of glycogen by a bi-functional mechanism. Each enzymatic activity of GDE is suggested to be associated with distinct domains; alpha-1,4-glycosyltransferase activity with the N-terminal domain and alpha-1,6-glucosidase activity with the C-terminal domain. Here, we present the biochemical features of the GDE from Saccharomyces cerevisiae using the substrate glucose(n)-beta-cyclodextrin (Gn-beta-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1-644) showed alpha-1,4-transferase activity on maltotetraose (G4) and G4-beta-CD, yielding various lengths of (G)(n). Surprisingly, the N-terminal domain also exhibited alpha-1,6-glucosidase activity against G1-beta-CD and G4-beta-CD, producing G1 and beta-CD. Mutational analysis showed that residues D535 and E564 in the N-terminal domain are essential for the transferase activity but not for the glucosidase activity. These results indicate that the N-terminal domain (1-644) alone has both alpha-1,4-transferase and the alpha-1,6-glucosidase activities and suggest that the bi-functional activity in the N-domain may occur via one active site, as observed in some archaeal debranching enzymes. (C) 2014 Elsevier Inc. All rights reserved.