Regulation Of The OsHAL3 Protein Function By Sitedirected Mutagenesis Of Amino Acids

It has been shown that the OsHAL3 protein in rice has 4’-phosphopantothenoylcysteine decarboxylase（PPCDC）activity.The PPCDC is a key enzyme in the coenzyme A synthesis pathway and the OsHAL3protein has been shown to be involved in the salt tolerance of the rice.We hypothesized that the OsHAL3protein can achieve the resistance to salt stress by regulating the level of the coenzyme A in rice through its PPCDC function.In order to regulate the PPCDC activity of OsHAL3 protein,we mutated some amino acid sites and analyze if the sites are the key sites which affect PPCDC activity.We hope to eventually improve the salt tolerance of the rice by regulating the PPCDC activity of OsHAL3 protein.We constructed some expression vectors of the OsHAL3 gene with point mutation,which were transformed into the E.coliΔdfp strain.The temperature-sensitive assays and transformant growth rate assays showed:the single-mutations of some amino acids in OsHAL3 lead to the loss of the PPCDC function,which including Leu²⁵,Gly²⁹,Ala³²,Ala³³,Leu³⁹,Ala⁴⁷,Val⁴⁹,Arg⁵⁰,Leu⁹⁴,Trp⁹⁷,Asp⁹⁹,Ala¹⁰⁴,Ala¹¹⁵,Gly¹¹⁷,Val¹³⁸,Ala¹⁴¹,Met¹⁴²,Gly¹⁸²,Ile¹⁹⁰.Three single-mutations of Met¹⁸⁴ to Leu¹⁸⁴,Leu⁶⁶ to Ile⁶⁶,and Ala¹⁰⁸ to Trp¹⁰⁸ lead to enhanced protein PPCDC function.However,the mutations of Ile⁹²,Glu⁹³,Leu¹²²,Ala¹⁷⁵,and Gly¹⁷⁷ did not affect the PPCDC function of the OsHAL3 protein.We simulated the spatial structures of a series of mutated OsHAL3 proteins by homology modeling,and initially predicted that the substrate binding domain,PXMNXXMW domain and substrate recognition folder domain are the PPCDC active centers of the OsHAL3 protein.Ala³²,Ala³³,Lys³⁵,Leu³⁹,Pro¹⁴⁰,Ala¹⁴¹,Met¹⁴²,Pro¹⁶⁹,Cys¹⁷⁶,Gly¹⁸⁰,Gly¹⁸²,Met¹⁸⁴ are key amino acid sites of PPCDC active center of the OsHAL3 protein.We compared the spatial structure of the OsHAL3 protein and those of the mutated OsHAL3 proteins,and preliminarily speculated the reasons which caused the changes of the PPCDC activity of the OsHAL3protein.It may be that the change of some amino acid residue groups attenuates the steric hindrance within the active center and enhances the activity of PPCDC.On the contrary,the change of some amino acid residue groups enhances the internal tension of the molecule,increases the steric hindrance effect,and then causes the loss of PPCDC activity.The insignificant changes in the amino acid residue groups did not affect PPCDC activity.