Expression of the Potato Tuber ADP-glucose Pyrophosphorylase in Escherichia coli

A.A. IGLESIAS, G.F. BARRY, C. MEYER, L. BLOKSBERG, P.A. NAKATA, T. GREENE, M.J. LAUGHLIN, T.W. OKITA, G. KISHORE, J. PREISS

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 1993 vol. 268 p. 1081 - 1086

0021-9258

cDNA clones encodingt he putative mature formosfthe large and small subuniotsf the potato tuberA DPglucosepyrophosphorylase have been expressed separatelyand together in an Escherichia coli B mutantdeficient in ADP-glucose pyrop~osphorylasea ctivity.Expression of both subunits from compatible vectorsresulted in restoration of ADP-glucose pyrophosphorylaseactivity. Maximal enzyme activity required bothsubunits. The expressed ADP-glucose pyrophosphorylasewas purified and characterized. The recombinantenzyme exhibited catalytica nd allosteric kinetic propertiesvery similar to the enzyme purified from potatotuber. The expressed enzyme activity was neutralizedby incubation with antibodies raised against potatotuber and spinach leaf ADP-glucose pyrophosphorylasesbut not with anti-Escherichia coli enzyme serum.3-Phosphoglycerate was the most efficient activatorand its effect was increased by dithiothreitol. In theADP-glucose synthesis direction, 3-phosphoglycerateactivated the recombinant enzyme nearly 100-fold inthe presenceo f dithiothreitol, with anA O.v~al ue of 57pM. The recombinant ADP-glucose pyrophosphorylasewas less sensitive toP i inhibition and more sensitive toheat denaturation than the potato tuber enzyme. Resultssuggest that bacterial expression of potato tubereDNAs could bc used to study the role and interactionof the subunits of the native ADP-glucose pyrophosphorylase.