Functional characterization of alpha-glucan,water dikinase, the starch phosphorylating enzyme.
GWD (alpha-glucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of alpha-1,6 branch points in its polyglucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs.
Mikkelsen
René
R
Center for Molecular Plant Physiology (PlaCe), Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Baunsgaard
Lone
L
Blennow
Andreas
A
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005936:Glucans / Q000378:metabolism*; D006639:Histidine / Q000032:analysis / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011198:Solanum tuberosum / Q000201:enzymology; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005936:Glucans; D010940:Plant Proteins; D006639:Histidine; D013213:Starch; D010770:Phosphotransferases