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. 1996 Feb 20;93(4):1509–1513. doi: 10.1073/pnas.93.4.1509

Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.

T W Greene 1, S E Chantler 1, M L Kahn 1, G F Barry 1, J Preiss 1, T W Okita 1
PMCID: PMC39970  PMID: 8643662

Abstract

ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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