Cloning and expression of the glgC gene from Agrobacterium tumefaciens: purification and characterization of the ADPglucose synthetase.
The gene encoding ADPglucose synthetase (EC 2.7.7.27) from Agrobacterium tumefaciens was isolated and expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity in steps including ion-exchange and hydrophobic chromatography. The same purification procedure was utilized to purify ADPglucose synthetase from A. tumefaciens cells. The enzymes from the two sources were purified and characterized and were found to have identical kinetic, regulatory, and structural properties. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only one polypeptide band of 50 kDa was detected. In immunoblotting following electrophoresis, the 50-kDa band reacted with antibodies raised against the Escherichia coli ADPglucose synthetase; there was no reaction with antibodies raised against the spinach enzyme. The immunoreactivity of the A. tumefaciens ADPglucose synthetase was confirmed in antibody neutralization assays. Using gel filtration, the native enzyme was shown to be a tetramer. Fructose 6-phosphate and pyruvate were the most effective activators of the enzyme; maximal activation was observed in the ADPglucose synthesis direction, in which the enzyme was activated about ninefold by fructose 6-phosphate and fivefold by pyruvate. Both activators increased the affinity of the enzyme for the substrates ATP and glucose 1-phosphate. Inorganic orthophospate, ADP, AMP, and pyridoxal phosphate behaved as inhibitors of the enzyme. The distinctive regulatory properties of the enzyme from A. tumefaciens are compared with those of two enterobacterial enzymes and discussed in the context of their deduced amino acid sequences.
Uttaro
A D
AD
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Ugalde
R A
RA
Preiss
J
J
Iglesias
A A
AA
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase; D008969:Molecular Sequence Data; D005810:Multigene Family; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase