Structural Studies Of Rice Auxin-Amido Synthetase GH3-8

Plant auxin is crucial for the growth and development of plants,and even affects the ripening of plant products.Therefore,it is particularly important to control the homeostasis of free auxin in plants.The GH3 family of auxin-amino acid synthetase plays a key role in maintaining auxin content.It catalyzes the conjugation of excess auxin with amino acids to maintain the steady state of free auxin in plant.Os GH3-8 is a kind of auxin-amino acid synthetase,which belongs to group II of the rice GH3 family.It has been shown that Os GH3-8protein is responsible for attaching Asp to IAA and Asp and participates in regulation of growth and development as well as basic immunity of plants.However,the mechanism of Os GH3-8 catalysis remains unclear.To better understand the molecular mechanisms of Os GH3-8 protein catalysis,we utilize the structural biology approach to obtain the three-dimensional crystal structure of this protein and perform structural analyses to explain its biochemical function.In this thesis,the preliminary analysis of the Os GH3-8 protein sequence through several bioinformatics methods,a reasonable truncation design to ensure the solubility and stability of this protein,and molecular cloning of the truncated target gene into p RSFDuet-1 for protein expression.Through a serial of purification experiments,the target protein is purified to the extent of crystallization quality.After addition of small molecule substrates into the protein solution,the protein-substrate complex is crystallized by the hanging drop vapor diffusion method.The crystal structure of a complex between Os GH3-8 and AMP was finally obtained by X-ray crystal diffraction and structure determination.Through structural analysis,we determined the binding site of AMP in the Os GH3-8 protein.Comparing the structure and sequence of Os GH3-8 with those of other reported GH3 proteins in plants,we found that the key amino acids of the ATP/AMP binding site are highly conserved,which means that GH3 proteins in a dicotyledonous and monocotyledonous plants may have similar ATP/AMP binding mode.Compared with the structure of At GH3-5with IAA,no changes in the orientation of key amino acids were found.Therefore,Os GH3-8 and At GH3-5 may bind IAA in a similar mode.To the best of our knowledge,this is the first GH3 protein crystal structure determined from monocotyledonous plants.Our work promotes further understanding of the substrate binding mode and catalytic mechanism of Os GH3-8,which may provide theoretical instructions and molecular materials to coordinate growth and stress resistance in crop plants.