Characterisation of disproportionating enzyme from wheat endosperm

Area of Science:

• Biochemistry
• Plant Science
• Enzymology

Background:

• Disproportionating enzyme (D-enzyme), an alpha-1,4 glucanotransferase, modifies oligosaccharide chain lengths.
• D-enzyme is well-characterized in plants like potato and Arabidopsis.
• Its function and abundance in cereals, major starch sources, remain largely unknown.

Purpose of the Study:

• Investigate the abundance and function of D-enzyme in wheat (Triticum aestivum).
• Isolate and characterize D-enzyme cDNA clones from wheat.
• Confirm D-enzyme activity and localization in wheat.

Main Methods:

• Isolation of two putative D-enzyme cDNA clones (TaDPE1-e and TaDPE1-l) from wheat and Aegilops taushii endosperm and leaf cDNA libraries, respectively.
• Analysis of deduced polypeptide sequences for identity with known D-enzymes and conserved residues.
• Protein analysis to determine D-enzyme presence in developing endosperm and germinating seeds.
• Partial purification of wheat endosperm D-enzyme and in vitro activity assays.

Main Results:

• Two wheat D-enzyme cDNA clones, TaDPE1-e and TaDPE1-l, were isolated, encoding polypeptides with high sequence identity (96%) and significant similarity to corn and rice D-enzymes.
• Mature polypeptides were calculated to be 59 and 60 kDa, containing expected conserved residues.
• Protein analysis confirmed D-enzyme presence in the amyloplast of developing endosperm and in germinating seeds.
• Partially purified D-enzyme exhibited disproportionating activity in vitro, cleaving maltotriose to glucose and utilizing maltoheptaose.

Conclusions:

• D-enzyme is present and functional in wheat, specifically in developing endosperm and germinating seeds.
• The isolated wheat D-enzyme sequences are homologous to those in other plant species.
• This study provides foundational insights into the role of D-enzyme in cereal starch metabolism.