Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme

作者：Min ho Lee; Hyung Nam Song; Ji Eun Cho; Tran Phuong Lan; Sunghoon Park; Jong Tae Park; Eui Jeon Woo^*

来源：Biochemical and Biophysical Research Communications, 2014, 445(1): 107-112.

DOI：10.1016/j.bbrc.2014.01.134

• Saccharomyces cerevisiae
• Glycogen debranching enzyme (GDE)
• alpha-1,4-Transferase activity
• alpha-1,6-Glucosidase activity
• Bi-functional

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.

• 出版日期2014-2-28