Amylomaltase

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Introduction

Transitory starch in leaves is stored in the chloroplast during the day and then broken down for export at night. Several groups have investigated starch breakdown in intact chloroplasts isolated from spinach. Early experiments showed that in the presence of phosphate, the principal products of starch breakdown were triose phosphate and 3-phosphoglycerate. In the absence of phosphate, starch can also be converted to maltose and glucose. Stitt and Heldt (1981) showed that phosphorolytic starch degradation is the major pathway for starch breakdown. Data from other groups suggest that both phosphorolytic and hydrolytic starch degradation are important.

Servaites and Geiger (2002) found that glucose, maltose, and isomaltose were the major products mobilization exported from chloroplast at night, while Ritte and Raschke (2003) showed that starch breakdown in the light in guard cell chloroplasts results in a large export of maltose. Weiser et al. (2003) found that the major products exported were maltose and glucose within 1-3 hours after the lights were turned out, excluding isomaltose.  The synthesis of sucrose is the major fate for the carbon released by transitory starch degradation. A large amount of maltose is exported from chloroplasts, indicating that there must be some enzymes in the cytoplasm that metabolize maltose to precursors for sucrose synthesis.

Amylomaltase is a type II 4-α-glucanotransferase (GTase; EC 2.4.1.25) that catalyzes the intermolecular glucan transfer reaction from one 1,4-α-glucan molecule to another, or to glucose. Amylomaltase was first discovered in Escherichia coli and its gene (malQ) was cloned from Streptococcus pneumoniae, Escherichia coli, Thermus aquaticus and Clostridium butyricum. Boos and Shuman (1998) showed that maltose and maltodextrin are catabolized to glucose and G1P by amylomaltase (MalQ), maltodextrin phosphorylase (MalP) and maltodextrin glucosidase (MalZ).

The D-enzyme

A similar type II 4-a-GTase, disproportionating-enzyme (D-enzyme), also exists in plants. D-enzymes were first discovered in potato tubers and subsequently found in many plant organs. By knocking out the plastidic D-enzyme, the majority of GTase activity was found in the chloroplast. The plastidic D-enzymes are responsible for converting short oligosaccharides into longer chains to provide substrates for plastidic starch phosphorylase and β-amylase. Arabidopsis plants lacking the plastidic D-enzyme (dpe1-1) cause accumulation of maltotriose-maltoheptaose but not maltose.

After the Arabidopsis genome sequence was completed in 2000, another gene predicted to encode 4-α- GTase was found on chromosome 2 (At2g40840). This gene does not appear to encode any targeting sequence, as judged by Target-P, and the enzyme is predicted to be present in the cytosol. To investigate the function of this enzyme, two T-DNA knockout mutants of Arabidopsis thaliana (L) Heynh. were identified that completely lack the cytosolic amylomaltase (DPE2) transcript, and proposed that maltose metabolism in the cytosol of Arabidopsis leaves is similar to maltose metabolism in the cytoplasm of E. coli.

Figure 1. Structure of the dpe2-1 and dpe2-2 loci (Lu, Y.; Sharkey, T.D. 2004)

Discussion

The nature of the cytosolic polysaccharides involved in this metabolism in Arabidopsis is unknown, but the ability of heteroglycans to initiate phosphorylase reactions appears to be important. Advantages of having a cytosolic polysaccharide pool in the cytosol may include: (1) As an overflow for carbohydrates (such as maltose and glucose) formed from starch degradation at night when the amount of hexose exceeds the demand and transport of sucrose, acting as a soluble starch pool in the cytosol. (2) By forming polysaccharides, the molarity concentration of total sugars in the cytosol is maintained at a low level, so that the cells maintain an intracellular osmotic pressure that is beneficial to their overall metabolic reactions in the cytosol.

In conclusion, the DPE2 gene is required for maltose metabolism in Arabidopsis. Maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in E. coli. If the maltose/maltodextrin system of maltose metabolism is the primary mechanism for the conversion of carbon in starch to sucrose, the regulation of starch metabolism may be more easily understood as the regulatory mechanisms of the enzymes involved are elucidated.

Figure 2. Hypothetical pathway for transitory-starch breakdown and conversion to sucrose (Lu, Y.; Sharkey, T.D. 2004)