Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme.
Glycogen debranching enzyme (GDE) in mammals and yeast exhibits α-1,4-transferase and α-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; α-1,4-glycosyltransferase activity with the N-terminal domain and α-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)-β-cyclodextrin (Gn-β-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1-644) showed α-1,4-transferase activity on maltotetraose (G4) and G4-β-CD, yielding various lengths of (G)n. Surprisingly, the N-terminal domain also exhibited α-1,6-glucosidase activity against G1-β-CD and G4-β-CD, producing G1 and β-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 α-1,4-transferase and the α-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.
Department of Bio-analytical Science, University of Science and Technology, Daejeon 305-333, Republic of Korea; Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
Lee
L
Min-Ho
Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
Song
S
Hyung-Nam
Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
Cho
C
Ji-Eun
Department of Foodservice Management and Nutrition, College of Natural Science, Sangmyung University, Seoul 110-743, Republic of Korea.
Tran Phuong
TP
Lan
Purdue University, Department of Medicinal Chemistry and Molecular Pharmacology, 575 Stadium Mall Drive, West Lafayette, IN 47907, United States.
Park
P
Sunghoon
Department of Food Science and Technology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea.
Park
P
Jong-Tae
Department of Bio-analytical Science, University of Science and Technology, Daejeon 305-333, Republic of Korea; Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea. Electronic address: ejwoo@kribb.re.kr.
Woo
W
Eui-Jeon
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism*; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D003505:Cyclodextrins; D006004:Glycogen Debranching Enzyme System; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D008320:Maltose; C009819:maltotetraose; D016695:Glycosyltransferases; D005959:Glucosidases; D005947:Glucose
Bi-functional; Glycogen debranching enzyme (GDE); Saccharomyces cerevisiae; α-1,4-Transferase activity; α-1,6-Glucosidase activity