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Mutagenesis of the Potato ADPGlucose Pyrophosphorylase and Characterization of an Allosteric Mutant Defective in 3-Phosphoglycerate Activation
Thomas W. Greene, Sue Ellen Chantler, Michael L. Kahn, Gerard F. Barry, Jack Preiss and Thomas W. Okita
Proceedings of the National Academy of Sciences of the United States of America
Vol. 93, No. 4 (Feb. 20, 1996), pp. 1509-1513
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/38612
Page Count: 5
You can always find the topics here!Topics: Enzymes, Glycogen, Mutagenesis, Tubers, Complementary DNA, Genetic mutation, Biosynthesis, Enzyme activity, Biochemistry, Phosphates
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ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:α -D-glucose-1-phosphate adenylyltransferase, EC 18.104.22.168) catalyzes a key regulatory step in α -glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.
Proceedings of the National Academy of Sciences of the United States of America © 1996 National Academy of Sciences