Plant α-Glucosidase: Molecular Analysis of Rice α-Glucosidase and Degradation Mechanism of Starch Granules in Germination Stage

Abstract

In germination of plant seeds, storage starch is principally degraded by the combination of amylolytic enzymes. As starch is an insoluble granule, a conventional view of the degradation pathway is that the initial attack is performed by α-amylase having the starch granule-binding ability. Plant α-glucosidase was also capable of adsorbing and hydrolyzing starch granules directly, indicating a possible second pathway: the direct liberation of glucose from starch granules by plant α-glucosidase rather than the α-amylase-mediated system. We found that the starch-binding site of plant α-glucosidase was situated in its C-terminal region, of which function was independent of the catalytic domain. Site-directed mutagenesis analysis on the aromatic amino acid residues conserved in this region revealed that Trp803 and Phe895 of rice α-glucosidase were responsible for binding to starch granules. Mold α-glucosidases were devoid of the ability to attack starch granules. In plant seeds, multiple α-glucosidases have been observed. Two types of α-glucosidases, insoluble and soluble enzymes, were found in the germinating stage of rice. Expression patterns of their activities classified 14 rice varieties into two groups (Groups 1 and 2). In Group 1 varieties, insoluble enzyme decreased immediately after germination. The soluble enzyme increased by de novo synthesis. Group 2 maintained a constant activity level of insoluble and soluble α-glucosidases in germination. From Groups 1 and 2, we selected varieties of Akamai and Nipponbare, respectively, of which analysis elucidated interesting molecular mechanisms of insoluble and soluble enzymes: i) isoform and isozyme formations by post-translational proteolysis as well as by chromosomal gene expression; ii) characterization of purified enzymes exhibiting different activities to starch granules.