Discovery of the archaeal chemical link between glycogen (starch) synthase families using a new mass spectrometry assay.
Starch and its analogue glycogen are biosynthesized by enzymes that have been classified by sequence similarities into two families that have no significant sequence overlap: the animal/fungal glycogen synthases and the plant/bacterial glycogen (starch) synthases. Recent gene sequence analysis of putative archaea enzymes implicates them as a third family that links the structural and functional features of the other two classes. Herein, we present the first rapid electrospray ionization mass spectrometry-based assay to quantify any carbohydrate-polymerizing activity, the first cloning and recombinant expression as well as verification of the putative function of a glycogen synthase from the hyperthermophilic archaea Pyrococcus furiosus, and the characterization of a variety of glycogen synthases with the new assay. The new assay allowed the determination of Km and Vmax values for the rabbit, yeast, and P. furiosus glycogen synthases. Most surprisingly, unlike the synthases from rabbit or yeast and in contradiction to what would be expected from structural studies of other nucleotide-sugar binding proteins, the synthase from the archaea source accepts both uridine- and adenine-diphosphate activated glucose competitively and with comparable affinities to form a glucose polymer. This loose substrate specificity implicates this protein as the chemical link between the two branches of glycogen synthases that have evolved to accept primarily one or the other nucleotide as well as a good source enzyme for polymer bioengineering efforts.
Zea
Corbin J
CJ
Department of Chemistry and the Plant Sciences Institute, Gilman Hall, Iowa State University, Ames, Iowa 50011-3111, USA.
MacDonell
Stephen W
SW
Pohl
Nicola L
NL
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D006006:Glycogen Synthase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D020027:Pyrococcus furiosus / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D019843:Archaeal Proteins; D000245:Adenosine Diphosphate Glucose; D006006:Glycogen Synthase; D014532:Uridine Diphosphate Glucose