Characterisation of disproportionating enzyme from wheat endosperm

Abstract

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.

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

“…4) confirmed that the purified disproportionating enzyme was a typical DPE1. [12][13][14] Our results indicate that DPE1 is not significantly inhibited by acarbose, but can use it as donor substrate. This is the first example of an acarbosemodifying enzyme in GH family 77.…”

“…The transferring unit, exceptionally, is G3 when the donor substrate is G4. [12][13][14] We found a typical DPE1 in cotyledons of germinating adzuki beans, and the enzyme produced six compounds from acarbose and maltooligosaccharides. These six compounds were analyzed by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR).…”

Starch accumulation, activities of key enzyme and gene expression in starch synthesis of wheat endosperm with different starch contents

“…Starch has two major components, including amylose and amylopectin (Morell and Myers 2005;Tetlow 2006). Both amylose and amylopectin consist of a backbone of a-1,4 linked glucose residues, whereas amylose has a degree of polymerization (DP) of 1,000-5,000 and few branch points (fewer than 0.5% a-1,6 linkages), amylopectin is a very large molecule (DP 5,000-50,000) with frequent branches (3-4% a-1,6 linkages) (Bresolin et al 2006). Amylose, which makes up 20-30% of normal starch, is an essentially linear molecule.…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…Linear MOS also accumulate abnormally in the soluble fraction from the mutants. Analysis of D-enzyme levels in the developing endosperm of wheat is consistent with a role in starch biosynthesis for this enzyme [119]. However, the involvement of D-enzyme in amylopectin biosynthesis may vary depending on physiological conditions or tissue, as the effects of sta11 were only observed in conditions of nutrient starvation, not in exponential-phase cells.…”