Domain characterization of a 4-alpha-glucanotransferase essential for maltose metabolism in photosynthetic leaves.
Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-alpha-glucanotransferase (EC 2.4.1.25) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of approximately 150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the beta-anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.
Steichen
Jon M
JM
Departments of Botany and Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Petty
Ryan V
RV
Sharkey
Thomas D
TD
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry; D002855:Chromatography, Thin Layer / Q000379:methods; D005947:Glucose / Q000737:chemistry; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry*; D007700:Kinetics; D008320:Maltose / Q000737:chemistry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010788:Photosynthesis; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry
D002241:Carbohydrates; D006004:Glycogen Debranching Enzyme System; D013395:Sucrose; D008320:Maltose; C020569:4 alpha-glucanotransferase; D005947:Glucose