A novel type carbohydrate-binding module identified in alpha-glucan, water dikinases is specific for regulated plastidial starch metabolism.
The phosphorylation of the amylopectin fraction of starch catalyzed by the alpha-glucan, water dikinase (GWD, EC 2.7.9.4) plays a pivotal role in starch metabolism. Limited proteolysis of the potato tuber (Solanum tuberosum) GWD (StGWD, 155 kDa) by trypsin primarily produced stable fragments of 33 and 122 kDa, termed the SBD fragment and N11, respectively, as generated by trypsin cleavage at Arg-286. SBD and N11 were generated using recombinant DNA technology and purified to near homogeneity. Tandem repeat sequences, SBD-1 and SBD-2, of a region that is significantly similar in sequence to N-terminal regions of plastidial alpha-amylases are located in the N-terminus of StGWD. The SBD-1 motif is located within the sequence of the SBD fragment, and our results demonstrate that the fragment composes a new and novel carbohydrate-binding module (CBM), apparently specific for plastidial alpha-glucan degradation. By mutational analyses of conserved Trp residues located within the SBD-1 motif, W62 and W117, we show that these aromatic residues are vital for carbohydrate binding. N11 still possessed starch phosphorylating activity, but with a 2-fold higher specific activity compared to that of wild type (WT) StGWD using potato starch as the glucan substrate, whereas it had double the K(m) value for the same substrate. Furthermore, investigation of the chains phosphorylated by WT StGWD and N11 shows that N11 exhibits a higher preference for phosphorylating shorter chains of the amylopectin molecule as compared to WT. From analyses of the glucan substrate specificity, we found up to 5-fold higher specific activity for N11 using amylose as the substrate.
Mikkelsen
René
R
Center for Molecular Plant Physiology, Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, 1876 Frederiksberg C, Copenhagen, Denmark.
Suszkiewicz
Ken
K
Blennow
Andreas
A
D010446:Peptide Fragments / Q000378:metabolism; D017866:Phosphotransferases (Paired Acceptors) / Q000737:chemistry* / Q000235:genetics; D018087:Plastids / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D011198:Solanum tuberosum / Q000201:enzymology; D013213:Starch / Q000378:metabolism*
D010446:Peptide Fragments; D013213:Starch; D017866:Phosphotransferases (Paired Acceptors); C498198:alpha-glucan, water dikinase, Solanum tuberosum