Exploring The Difference Mechanism Of Nitrogen Fixation Ability Of Glutamate Dehydrogenase In Rice

Glutamate dehydrogenase(GDH)is an important enzyme to maintain the balance of carbon and nitrogen metabolism.GDH widely exists in plants,animals and microorganisms.According to the classification of cofactors,GDH can be divided into three types:NADPH-GDH type,NADH-GDH type and NADPH/NADH type.The introduction of GDH into rice by genetic engineering can obtain stable plant lines which can grow normally in nitrogen deficient soil.However,the food safety of transgenic rice is controversial.Among the four GDH expressed in rice,OsGDH4 was related to the ability of nitrogen fixation and belonged to NADPH-GDH type as the GDH expressed in fungi,but its ability of nitrogen fixation was weaker than that expressed in fungi.The purpose of this study is to study the nitrogen fixation mechanism of OsGDH4from the protein molecular structure level,in order to improve the endogenous nitrogen fixation ability of OsGDH4 in rice.Based on the known crystal structure 5xvx of Aspergillus niger GDH,we used Modeller software to model the homology of unknown fungal GDH and rice OsGDH4,and obtained the monomeric structures of nine fungal GDH and rice OsGDH4.The complex of GDH with ligand molecule AKG and cofactor NADPH was simulated by 2ns molecular dynamics,and the MM/PBSA binding energy was calculated.The correlation between the binding energy of GDH with AKG and NADPH and the K_mvalue of ammonia ion related to enzyme activity was analyzed.It is found that the binding energy of GDH with AKG and NADPH is positively correlated with K_m.By comparing and observing the structures of rice OsGDH4 and fungus GDH,we found that there were differences in the two key amino acids in their active centers.Primer sequence of OsGDH4 was designed to obtain the expression vector of OsGDH4containing p ET-32a using Eco RI and Kpn I sites.We designed two single mutants and a double mutant of OsGDH4 for the purpose to determine their experimental enzyme activity via enzymatic activity assay.In this study,the amino acids causing the difference of GDH between rice and fungi were identified by structural biology and computational biology.From the perspective of molecular biology,the experiment of rice OsGDH4 enzyme activity was designed.This provides an experimental basis for improving the nitrogen fixation ability of rice OsGDH4 from the perspective of amino acid mutation.It also provides a new strategy to improve nitrogen fixation ability of rice.