Expression and Characterization of Rice Disproportionating Enzymes

Abstract

Disproportionating enzyme (D-enzyme) is a 4-αglucanotransferase (EC 2.4.1.25) that cleaves an α-1,4 glucosidic bond of α-1,4 glucan (donor molecule) and transfers the resulting glucan moiety to a non-reducing end of an acceptor 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 1 7) and Arabidopsis, 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 amylomaltases 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 glucosyl 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 Arabidopsis 14 16) and potato. 17) Despite their similarity in primary 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 6.0 7.0, respectively. The optimum reaction temperature was 30 C for OsDPE1 and 39 C 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.

Plastidial Disproportionating Enzyme Participates in Starch Synthesis in Rice Endosperm by Transferring Maltooligosyl Groups from Amylose and Amylopectin to Amylopectin

“…We showed that DPE1 is expressed at a high level, specifically in rice endosperm, and that the activity of DPE1 is substantially high throughout starch accumulation and endosperm development. The temporal and spatial profiles of DPE1 transcripts were consistent with two previous reports (Ohdan et al, 2005;Akdogan et al, 2011). Furthermore, in wheat endosperm, the level of DPE1 was high at 5 to 10 DAF and higher in the later stages of seed development (Bresolin et al, 2006).…”

“…The activity of DPE1 in transgenic rice seeds was further analyzed by its activity of releasing Glc from maltotriose (Akdogan et al, 2011). The OX and Ri lines tested exhibited significantly higher and lower DPE1 activities than did the wild type, respectively.…”

“…The homogenate was centrifuged (20,000g for 10 min at 4°C), and the supernatant was used for the assay of DPE1. DPE1 activity was assayed by a method adapted from Akdogan et al (2011). Enzyme extract (50 mL) was mixed with 50 mL of 20 mM maltotriose.…”

“…Circumstantial evidence also suggests the involvement of DPE1 in storage starch synthesis in cereal crop endosperm. In rice, the transcriptional level of DPE1 is high in developing endosperm (Ohdan et al, 2005;Akdogan et al, 2011), and in wheat, DPE1 activity and protein level are coordinated with production of endosperm starch (Bresolin et al, 2006).…”

“…The rice genome contains at least 27 genes encoding starch-biosynthesizing enzymes: six for AGPase, two for GBSSI, eight for SS, three for BE, four for DBE, two for Pho, and two for D-enzyme (Ohdan et al, 2005). Among them, the functions of the former six groups have been studied intensively; however, little is known about the precise role of DPE1 in starch metabolism in developing rice endosperm, although the expression profile of rice DPE1 and enzymatic properties of recombinant rice DPE1 have been examined (Ohdan et al, 2005;Akdogan et al, 2011). Here, we report the functional characterization of DPE1 in rice storage starch synthesis.…”