| Starch is the major higher plant storage carbohydrate. It is reported that the starch degradation in chloroplast is closely related with phophatase. As a member of DSPs family, DSP4 is one of the most important phosphatase binding to the surface of starch granules in chloroplast. It contains a PTP and a starch binding domain (SBD). The major function of PTP is dephopharylate substrate, while SBD mainly binds to starch. To date, DSP4 is the only enzyme in DSPs which could affect the morphology of starch granules directly. The former researches have showed that starch in WT is flat, while dsp4 starch granules are round and have larger diameter. Furthermore, the starch level in dsp4 is 2.5-4 folds higher than WT. However, little is known about the structure and properties of DSP4, especially DSP4 SBD, including which how amino acids are involved in binding to starch and how it regulates the starch degradation.A BLAST search was performed in TAIR,PDB databases using the sequence of DSP4. The ClustalⅩresult showed the structure of GBD of AMPK (32 (PDB ID:2F15) shared the highest similarity with DSP4 SBD. Based on the solved structure of 2F15, a 3D model of DSP4 SBD was built using MODELLER 9v6. Furthermore, Webfeature was used here to predict the potential starch and calcium binding sites in the model. DSP4 SBD model takes the C2-domain fold which contains a Greek-key motif. The striking feature of this structure lies in the 3 carbohydrate-binding regions (CBR1-3). CBR3 contained 2 key residues (D311, E313) closely related to starch binding, besides, it was involved in forming one of the calcium binding sites—Site I, which contained D311 and E313. In vitro, Site-directed mutageneses were performed to D311 and E313 of CBR3, E305A, D328A and W314S were used as controls here. All proteins were successfully expressed and purified in E.coli. Purified wild type and mutant DSP4 proteins were grouped into two. One was incubated with starch directly. Western blot analysis showed that the starch binding capacity of mutant E305A wasn't affected, while the enzyme activity of mutant D328A was partly lower compared to wild type DSP4. And mutant D311A and E313A lost the starch binding capacity completely as mutant W314S, which indicates residues D311 and E313 were key to keep the starch binding capacity of DSP4. When extra 5mM Ca2+ was added to the incubation buffer, mutants DSP4 regained the starch binding function. It indicated that calcium was important to DSP4. In vivo, mutants DSP4 was co-expressed with GFP in Arabidopsis, and plants GFP::D311A/dsp4, GFP::E313A/dsp4 and GFP::W314S/dsp4 were constructed. GFP::DSP4/dsp4 was used as control here. Fluorescence microscope results showed that bright GFP fluorescence could be examined from the starch granules extracted from GFP::DSP4ldsp4. While no obvious GFP could be seen in the starch granules of GFP::D311A/dsp4, GFP::E313A/dsp4 and GFP::W314S/dsp4. This was consistent with the In vitro results, and had further verified the importance of residues D311 and E313 in DSP4. SEM checked the morphology of the starch granules in wild type and mutants DSP4. No obvious difference could be observed about the morphology of starch granules between wild type and mutants. SEM showed that they were all flat and with relative small diameters. This was different from the starch granules in dsp4, which was round and big. This indicated that amino acids D311 and E313 had no direct connection with the starch morphology control in DSP4 sequence. The present study has deepened our recognition towards the structure and function of DSP4, which laid a basis for elaborating the course of starch degradation.
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