Background:
Arabidopsis maltodextrin disproportionating enzyme 1 (AtDPE1) plays a key role in chloroplast starch degradation.Results: Six AtDPE1 structures define the active site and reveal mechanistically relevant conformations of both the enzyme and substrate.Conclusion: Substrates are captured through loop rearrangements; the subtle deployment of active site residues controls catalysis.Significance: A molecular level understanding of the complete disproportionation cycle of AtDPE1 is presented.
4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1,4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1,4-α-D-glucan to an acceptor mono-or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6-8 and 37 °C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16-50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP>60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.
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