title: Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis.
abstract: Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase). After each round of mutagenesis, iodine staining and antibody capture activity assays at varying temperatures were used to select the optimum positions and amino acid changes for the next rounds of mutagenesis. After three iterations, the signals from whole-colony iodine staining were saturated and a heat stable AGPase variant was obtained. Kinetic studies of the heat stable mutant showed that it also had an unexpected increased affinity for the activator, 3-PGA. This is particularly valuable as both the temperature stability and allosteric properties of AGPase significantly influence grain yield.
authors.affiliation: Program in Plant Molecular and Cellular Biology, P.O. Box 110690, 1253 Fifield Hall, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Boehlein
authors.affiliation: Program in Plant Molecular and Cellular Biology, P.O. Box 110690, 1253 Fifield Hall, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Janine R
authors.initials: JR
authors.lastname: Shaw
authors.affiliation: 127 Chemistry Research Building, Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Jon D
authors.initials: JD
authors.lastname: Stewart
authors.affiliation: 127 Chemistry Research Building, Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Bradford
authors.initials: B
authors.lastname: Sullivan
authors.affiliation: Program in Plant Molecular and Cellular Biology, P.O. Box 110690, 1253 Fifield Hall, University of Florida, Gainesville, FL 32611, USA. Electronic address: lchannah@ufl.edu.
authors.forename: L Curtis
authors.initials: LC
authors.lastname: Hannah
mesh_terms: D018360:Crystallography, X-Ray; D056625:Endosperm / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D018506:Gene Expression Regulation, Plant; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D003313:Zea mays / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D051599:Glucose-1-Phosphate Adenylyltransferase
keywords: ADP-glucose pyrophosphorylase; Grain yield; Heat stability; Iterative saturation mutagenesis; Starch biosynthesis