Cloning and Expression of the glgC Gene from Agrobacterium tumefaciens. Purification and Characterization of the ADPglucose Synthetase

A.D. UTTARO, R.A. UGALDE, J. PREISS Y A.A. IGLESIAS

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Academic Press

Año: 1998 vol. 357 p. 13 - 21

0003-9861

The gene encoding ADPglucose synthetase (EC2.7.7.27) from Agrobacterium tumefaciens was isolatedand expressed in Escherichia coli. The recombinantprotein was purified to electrophoretic homogeneityin steps including ion-exchange and hydrophobicchromatography. The same purification procedurewas utilized to purify ADPglucose synthetase from A.tumefaciens cells. The enzymes from the two sourceswere purified and characterized and were found tohave identical kinetic, regulatory, and structuralproperties. In polyacrylamide gel electrophoresis inthe presence of sodium dodecyl sulfate, only onepolypeptide band of 50 kDa was detected. In immunoblottingfollowing electrophoresis, the 50-kDa bandreacted with antibodies raised against the Escherichiacoli ADPglucose synthetase; there was no reactionwith antibodies raised against the spinach enzyme.The immunoreactivity of the A. tumefaciens ADPglucosesynthetase was confirmed in antibody neutralizationassays.Using gel filtration, the native enzyme was shown tobe a tetramer. Fructose 6-phosphate and pyruvatewere the most effective activators of the enzyme; maximalactivation was observed in the ADPglucose synthesisdirection, in which the enzyme was activatedabout ninefold by fructose 6-phosphate and fivefold bypyruvate. Both activators increased the affinity of theenzyme for the substrates ATP and glucose 1-phosphate.Inorganic orthophospate, ADP, AMP, and pyridoxalphosphate behaved as inhibitors of the enzyme.