کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1925010 | 1536334 | 2015 | 10 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis](/preview/png/1925010.png)
• ADP-glucose pyrophosphorylase is thermo-sensitive and linked to grain loss.
• Three rounds of iterative saturation mutagenesis was employed.
• We obtained an enzyme with higher heat stability and lower Ka for the activator.
• A link between temperature stability and allostery was shown.
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.
Journal: Archives of Biochemistry and Biophysics - Volume 568, 15 February 2015, Pages 28–37