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Free Content A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits

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Summary

The heterotetrameric, allosterically regulated enzyme, adenosine-5′-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.
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Keywords: ADP-glucose pyrophosphorylase; bacterial expression; maize endosperm; potato tuber; starch synthesis; subunit interactions

Document Type: Research Article

Affiliations: 1: Program in Plant Molecular and Cellular Biology and Horticultural Sciences, University of Florida, PO Box 110690, 2211 Fifield Hall, Gainesville, FL 32611, USA 2: Biochemistry and Molecular Biology, University of Florida, PO Box 100245, R4-146 ARB, Gainesville, FL 32610-0245, USA 3: Dow Agroscience LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA 4: Program in Plant Molecular and Cellular Biology and Microbiology and Cell Science, University of Florida, PO Box 110700, 1052 Microbiology and Cell Science Building, Gainesville, FL 32611-0700, USA 5: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA

Publication date: February 1, 2005

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