Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli.

cDNA clones encoding the putative mature forms of the large and small subunits of the potato tuber ADPglucose pyrophosphorylase have been expressed separately and together in an Escherichia coli B mutant deficient in ADP-glucose pyrop~osphorylase activity.Expression of both subunits from compatible vectors resulted in restoration of ADP-glucose pyrophosphorylase activity.Maximal enzyme activity required both subunits.The expressed ADP-glucose pyrophosphorylase was purified and characterized.The recombinant enzyme exhibited catalytic and allosteric kinetic properties very similar to the enzyme purified from potato tuber.The expressed enzyme activity was neutralized by incubation with antibodies raised against potato tuber and spinach leaf ADP-glucose pyrophosphorylases but not with anti-Escherichia coli enzyme serum.3-Phosphoglycerate was the most efficient activator and its effect was increased by dithiothreitol.In the ADP-glucose synthesis direction, 3-phosphoglycerate activated the recombinant enzyme nearly 100-fold in the presence of dithiothreitol, with an A O .~ value of 57 pM.The recombinant ADP-glucose pyrophosphorylase was less sensitive to Pi inhibition and more sensitive to heat denaturation than the potato tuber enzyme.Results suggest that bacterial expression of potato tuber eDNAs could bc used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.ADP-glucose (ADP-Glc)' pyrophosphorylase (ATPwglucose-1-phosphate adenylyltransferase, EC 2.7.7.27) plays a pivotal role in the biosynthesis of glycogen and starch in bacteria and plants, respectively (1,2).This enzyme mediates the synthesis of ADP-Glc and PPi from Glc-1-P and ATP, and the product, ADP-Glc, serves as the activated glucosyl donor in a-1,4-glucan synthesis.The catalytic activity of both the bacterial and plant enzymes are subject to allosteric