Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity.
Glycogenin, the self-glucosylating primer for glycogen synthesis, is expressed in wild-type E. coli as a recombinant protein in an already partly glucosylated form, owing to the presence of its substrate, UDP-glucose. By using an E. coli mutant strain lacking in UDP-glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate-free glycogenin (apo-glycogenin) in good yield. When provided with UDPxylose, it autocatalytically adds 1 xylose residue. With UDP-glucose, an average of 8 glucose residues are added. However, release of the self-synthesized maltosaccharide chains with isoamylase reveals them to be a mixture. Chains as long as 11 glucose residues (maltoundecaose) are present. The ability of recombinant apo-glycogenin to self-glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr-194 of the protein.
Alonso
M D
MD
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, FL 33101.
Lomako
J
J
Lomako
W M
WM
Whelan
W J
WJ
Preiss
J
J
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005947:Glucose / Q000032:analysis; D005964:Glucosyltransferases; D006003:Glycogen / Q000096:biosynthesis; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D009154:Mutation / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism
D006023:Glycoproteins; D011994:Recombinant Proteins; C051887:glycogenin; D014540:Uridine Diphosphate Xylose; D006003:Glycogen; D005964:Glucosyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D005947:Glucose; D014532:Uridine Diphosphate Glucose