Characterisation of disproportionating enzyme from wheat endosperm.
Disproportionating enzyme or D-enzyme (EC 2.4.1.25) is an alpha-1,4 glucanotransferase which catalyses cleavage and transfer reactions involving alpha-1,4 linked glucans altering (disproportionating) the chain length distribution of pools of oligosaccharides. While D-enzyme has been well characterised in some plants, e.g. potato and Arabidopsis, very little is known about its abundance and function in cereals which constitute the major source of starch worldwide. To address this we have investigated D-enzyme in wheat (Triticum aestivum). Two putative D-enzyme cDNA clones have been isolated from tissue-specific cDNA libraries. TaDPE1-e, from an endosperm cDNA library, encodes a putative polypeptide of 575 amino acid residues including a predicted transit peptide of 41 amino acids. The second cDNA clone, TaDPE1-l, from an Aegilops taushii leaf cDNA library, encodes a putative polypeptide of 579 amino acids including a predicted transit peptide of 45 amino acids. The mature polypeptides TaDPE1-e and TaDPE1-l were calculated to be 59 and 60 kDa, respectively, and had 96% identity. The putative polypeptides had significant identity with deduced D-enzyme sequences from corn and rice, and all the expected conserved residues were present. Protein analysis revealed that D-enzyme is present in the amyloplast of developing endosperm and in the germinating seeds. D-enzyme was partially purified from wheat endosperm and shown to exhibit disproportionating activity in vitro by cleaving maltotriose to produce glucose as well as being able to use maltoheptaose as the donor for the addition of glucans to the outer chains of glycogen and amylopectin.
Bresolin
Nicole S
NS
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.
Li
Zhongyi
Z
Kosar-Hashemi
Behjat
B
Tetlow
Ian J
IJ
Chatterjee
Manash
M
Rahman
Sadequr
S
Morell
Matthew K
MK
Howitt
Crispin A
CA
D000595:Amino Acid Sequence; D000687:Amylopectin / Q000378:metabolism; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000032:analysis; D018628:Gene Dosage; D005936:Glucans / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D006109:Poaceae / Q000201:enzymology / Q000235:genetics; D011123:Polyploidy; D012639:Seeds / Q000201:enzymology*; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism; D014312:Trisaccharides / Q000378:metabolism; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D005936:Glucans; D006004:Glycogen Debranching Enzyme System; D014312:Trisaccharides; C031146:maltoheptaose; C008317:maltotriose; D013213:Starch; D006003:Glycogen; D000687:Amylopectin; C020569:4 alpha-glucanotransferase