Structural Insight Into Arabidopsis Protein GPRAT2

Glutamine Phosphoribosylpyrophosphate Amidotransferase,GPRAT for short,it is the key enzyme of the first step in the synthesis of purine nucleotides.GPRAT2 is one of the three isozymes of GPRAT.A phenyltriazole acetic acid compound DAS734 can bleach dicotyledonous weeds by inhibiting the catalytic reaction of AtGPRAT2.DAS734 is a specific inhibitor to AtGPRAT2.It is important to study Arabidopsis thaliana GPRAT2 and the mechanism of DAS734 and AtGPRAT2 by structural biology,it has great significance to understand the compounds regulating the metabolism of plants herbicides synthesis.In this study,we successfully cloned the gene AtGPRAT2 and it was properly expressed and purified in E.coli through His-tag purification methods.We got eleven conditions under which crystals can grow.Through precipitant,pH,additives optimization experiments,we obtained high resolution diffraction data using the crystals under the HR2-110-11#optimization condition.Moreover,we got 3.1 A diffraction data and finally the crystal structure of AtGPRAT2 has been resolved.DAS734 was successfully prepared by the synthesis method in the literature.In addition,we have conducted researches to obtain the structure of the protein complexes of AtGPRAT2 and DAS734 by co-crystallization methods.And there are two ways,one way is adding DAS734 to the protein,and the other way is adding DAS734 in antifreeze fluid before the X-ray diffraction,we designed the soaking time and concentration.Finally we received 24 sets of data,after we analysed data but there was no DAS734.Literature shows that AtGPRAT2 enzyme activity decreases with the increase of the concentration of DAS734,while the mutant R264K has no effect with DAS734 concentration changing.Therefore,R264K mutation of pET-22b-AtGPRAT2 was successful and the mutated plasmid was transferred to E.coli.Through expressing and purification,we obtaine protein and make foundation for the experiment of enzyme activity.