Structural and biochemical analyses of the disproportionating isozyme 2 (DPE2)
Plant Physiology
Final Report Abstract
Starch metabolism in plants involves multiple pathways for storing or metabolizing sugars. A key player in this complex metabolic network is the disproportionating isozyme 2 (DPE2; 4-α-glucanotransferase, EC 2.4.1.25). DPE2 metabolizes the starch-derived disaccharide β-maltose by mediating an intermolecular transglycosylation reaction between donor and acceptor glycan chains. The DPE2 monomer has a molecular weight of 109 kDa. However, DPE2 exists in various oligomeric states including a plant derived complex with an apparent molecular weight of more than 1,000 kDa (state I) and a smaller oligomeric state with a molecular weight of approximately 300 kDa (state II). The precise reaction mechanism of the crucial DPE2-facilitated step in starch degradation as well as the structural basis of DPE2 function and oligomerisation is poorly understood. The ultimate goal of our project was to analyse the structure of various oligomeric states of DPE2-complex in an integrative study combining single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Results of both structural studies would help us to understand the molecular mechanism of the key step in the carbon flux within the cytosol, and would shed a light into the reason for such a complex molecular architecture of DPE2. Purification protocols of state I and state II DPE2 by overexpression of recombinant DPE2 in E. coli had been well established during the preliminary phase of this project. However, after retirement of Prof. Steup in 2013 we could not re-establish the production / overexpression of recombinant DPE2 in the oligomeric state I in the new lab-environment (MPIMP-Golm or Charité). Despite our best efforts to improve and optimize our original protocol, purification of recombinant DPE2 only resulted in the dimeric state II. Therefore, we had to devise a new purification strategy and eventually managed to purify native DPE2 in the oligomeric state I from Arabidopsis thaliana plant. Another delay of our structural work was caused by severe technical problems with our high-end 300 kV Tecnai G2 Polara cryo-TEM. We obtained a solid, highly symmetric reconstruction corroborating the dodecameric state at intermediate resolution (16-19 Å) and got insight into intrinsic dynamic behavior of the complex. However, because the cryo-EM data were corrupted due to the long-lasting technical problems no high-resolution cryo-EM structure could be obtained so far and the work is still ongoing. Concerning solving the structure of dimeric, state II DPE2 by X-ray crystallography, we achieved considerable progress by reducing the surface entropy of DPE2 by directed mutagenesis. This strategy allowed us to improve the diffraction of crystals from 10 to below 6 Å, and opens possibility to improve it further by creating double and triple mutants, comprising mutations at 2 or even 3 high entropy regions. While unforeseen problems have not allowed us to finished the project in the originally proposed time, we have been able to overcome these problems and made considerable progress towards reaching our project goals. The work is still ongoing and recent data processing of new cryo-EM data collected on our finally fully repaired Polara cryo-TEM is promising. Thus, we are optimistic that we will be able to finish successfully the project in the near future.
Publications
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(2017) Photometric assay of maltose and maltoseforming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants. Anal Biochem 532: 72-82
Smirnova J, Fernie AR, Spahn CMT, Steup M
