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

Abstract

Starch is an abundant carbohydrate widespread in plants. This highenergy polysaccharide is preserved in the storage tissues, such as seeds. In the germination of plant seeds, the starch is degraded by several hydrolytic enzymes (αamylase, βamylase, debranching enzyme and αglucosidase), followed by conversion to the biological materials and energy necessary for growth. 1) In plant seeds, starch exists as practically insoluble polysaccharide called starch granules or raw starch. Therefore, the pathway of starch degradation in the germination stage has been generally considered as follows. 2) αAmylase is a key enzyme attacking the starch granules initially to liberate soluble dextrin. Produced dextrin is hydrolyzed by the further action combined with αamylase, βamylase and debranching enzyme to form oligosaccharides such as maltose. Finally, αglucosidase converts the oligosaccha-rides to glucose. Among these starch degrading enzymes αamylase has been considered to be the exclusive enzyme capable of hydrolyzing the starch granules, while the other enzymes were not. However, it had been reported that plant αglucosidases exhibited the ability to attack soluble starch effectively, 3,4) which suggested that α glucosidase hydrolyzed not only oligosaccharides such as maltose but starch as well in plant seeds. Recently, barley , 5,6) millet 7,8) and rice 9,10) αglucosidases were found to be capable of degrading the starch granules. The combination of αglucosidase and αamylase exhibited the syner-gism of degradation of starch granules. 5,6,11) It is of interest to learn the αglucosidasemediated starch metabolism in the germination stage. The first section of this article introduces our molecularlevel analysis on the degradation of starch granules catalyzed by plant αglucosidase. In the second section, we describe the multipleforma-tion mechanism of rice αglucosidases observed in the ripening and germination stages. αGlucosidases, which are synthesized in the ripening stage and preserved in dry seeds, are important in starch metabolism, since these enzymes hydrolyze starch granules before attacking by α amylase. Certain αglucosidase isozymes, which are expressed in the early stage of germination, also hydrolyze starch granules. It is valuable to learn the expression systems of αglucosidases in the dry seeds and germinating seeds. In both seeds, there were several enzymes, of which the expression feature differed by variety of rice, requiring more precise analysis to understand the contribution of each enzyme to starch metabolism. The second section of this article introduces the mechanism for the formation of rice αglucosidase isoforms and isozymes as well as their characteristics elucidated using purified en-Abstract: In germination of plant seeds, storage starch is principally degraded by the combination of amy-lolytic 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.