Expression and Characterization of Rice Disproportionating Enzymes

Abstract

Disproportionating enzyme (D-enzyme) is a 4-α-glucanotransferase (EC 2.4.1.25) that cleaves an α-1,4 glu-cosidic bond of α-1,4 glucan (donor molecule) and transfers the resulting glucan moiety to a non-reducing end of an ac-ceptor molecule by creating a new α-1,4 glucosidic bond. D-enzymes have been identifi ed in a number of plant species and characterized most extensively in potato 17) and Arabi-dopsis, 8,9) but also studied in pea, 10) wheat, 11) germinating barley seed 12) and sweet potato tubers. 13) These studies showed that plant D-enzymes have common reaction characteristics , where the smallest donor molecule is maltotriose, the smallest acceptor molecule is glucose and the smallest transferred glucan unit is maltose. Similar enzymes are also present in bacteria but bacterial 4-α-glucanotransferases are historically called amylomaltases; the term D-enzyme is only used for plant 4-α-glucanotransferases. Bacterial amy-lomaltases catalyze similar reactions, but differ slightly in substrate and reaction specifi city. 7) Amylomaltase can use maltose as a donor molecule (although maltose is less effective than larger maltooligosaccharides) and can catalyze glu-cosyl as well as glucanosyl transfer reactions. 7) It should be noted that reported plant D-enzymes cannot use maltose as a donor and never catalyze glucosyl transfers. The D-enzymes described above are now called DPE1 in order to distinguish them from recently identifi ed isoforms, DPE2, which have been characterized in both Arabidop-sis 1416) and potato. 17) Despite their similarity in primary structure, the reaction characteristics of DPE2 are completely different from those of DPE1. The only effective donor molecule for DPE2 appears to be maltose, and DPE2 cata-lyzes the transfer of a glucosyl unit from maltose to an acceptor molecule which may be glycogen or amylopec-tin 14,15) or a soluble heteroglycan. 16) From these reactions, DPE2 may need to be classifi ed as a 4-α-glucosyltransferase or transglucosidase, 18,19) instead of a glucanotransferase. The physiological functions of DPE1 and DPE2 in Arabi-dopsis have recently been determined. 9,14,15) These enzymes play very important roles in the process of converting transitory starch into sucrose at night. 19,20) In Arabidopsis leaf tissue , transitory starch in the chloroplast is degraded into maltose and maltotriose by chloroplastic β-amylase. DPE1 in chloroplasts converts maltotriose into glucose and malto-pentaose, which is then attacked by β-amylase to produce more maltose. Maltose thus produced within chloroplasts at night is exported into the cytosol through a newly identifi ed maltose transporter. 21) Cytosolic DPE2 is responsible for metabolizing this exported maltose, probably by transferring one glucose moiety of maltose to an unidentifi ed acceptor molecule. As has been described, both DPE1 and DPE2 play very important roles in Arabidopsis leaf tissue in the conversion of transitory starch into sucrose at night. However, the starch degradation pathway in other plant species and in other plant tissues may differ from that of Arabidopsis leaf tissue. 19,20) For example, in rice plants with reduced activity of chloro-plastic α-amylase, seed germination and seedling growth were delayed and showed starch-excess (sex) phenotype, 22) while α-amylase knockout mutants of Arabidopsis degraded their leaf starch normally. 23) On the other hand, repression Abstract: This work aims to characterize disproportionating enzyme (DPE1) and its isoform DPE2 in rice. Rice DPE genes (OsDPE1 and OsDPE2) were cloned and expressed in E. coli. The OsDPE1 and OsDPE2 genes encode proteins of 594 and 946 amino acids with a calculated molecular mass of 67 kDa and 108 kDa, respectively. Purifi ed recombinant OsDPE1 and OsDPE2 showed highest activity at around pH 7.0 and pH 6.07.0, respectively. The optimum reaction temperature was 30C for OsDPE1 and 39C for OsDPE2. Recombinant OsDPE1 disproportionates maltotriose to produce glucose and maltopentaose, and thus shares the defi ning behavior of D-enzymes. In our experiments, recombinant OsDPE2 catalyzed the glucose transfer reaction from maltose to an acceptor molecule such as glycogen. We also characterized the differences between the diurnal transcription profi les of OsDPE1 and OsDPE2 in rice leaves and seeds, and their temporal expression levels in developing rice seeds.