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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme^{1,[C],[W],[OA]}

Program in Plant Molecular and Cellular Biology and Horticultural Sciences, University of Florida, Gainesville, Florida 32610–0245

ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.

² Present address: Department of Biology, Pennsylvania State University, State College, Pennsylvania 16802.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: L. Curtis Hannah (hannah{at}mail.ifas.ufl.edu).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.138933

^* Corresponding author; e-mail hannah{at}mail.ifas.ufl.edu.

Received March 23, 2009; accepted July 17, 2009; published July 22, 2009.

This article has been cited by other articles:

				S. K. Boehlein, J. R. Shaw, L. C. Hannah, and J. D. Stewart Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase Plant Physiology, January 1, 2010; 152(1): 85 - 95. [Abstract] [Full Text] [PDF]