Research On The Key Amino Acids Of PPCDC Activity Of OsHAL3 Protein In Rice

OsHAL3 is a kind of flavoprotein in rice. It can serve as a 4’-phosphopantothenoylcysteinedecarboxyl-ase (PPCDC) and was involved in salt stress tolerance in rice.In order to further investigate key amino acid sites of the PPCDC activity of the OsHAL3 protein and reveal the molecular mechanisms of PPCDC catalytic reaction, we have produced a series of mutants of OsHAL3 including point mutations by PCR and constructed some vectors with different point mutations respectively in this study. Complementation test demonstrated that the OsHAL3 mutants with Ser65/Ala98/ Pro135/Glu9Gly163point mutation can rescue the temperature-sensitive of the E. coli △dfp strains which have a mutation in the dfp gene encoding a PPCDC.This suggests that the Glu9, Ser65,Ala98,Pro135,Gly163 of OsHAL3 protein are not key amino acid sites.And the different E.coli△dfp strains with OsHAL3 mutants carrying Ser28/Lys35/Pro105/Ile126/Ala129/Pro187point mutation can hardly grow at 42 ℃,which means that the conserved Ser28,Lys35,Pro105,Ile126, Ala129,Pro187residues of OsHAL3 are key active-site residues.The PPCDC activity of the mutant OsHAL3 proteins with Phe60/Pro140/Leu156/Pro169 mutation were partly reduced, which caused that the E. coli △dfp strains with above-mentioned OsHAL3 mutants grew slowly at 42℃. The E. coli △dfp strains with OsHAL3 Val184 grew faster than the positive control at 42℃,which means Val184 mutation can enhance the PPCDC function of the OsHAL3 protein.We predicted spatial structure of the OsHAL3 protein by SWISS-MODE and displayed spatial distribution of Pro169, Cys176, Gly180, Met184 amino acid residues in the protein OsHAL3 making use of Deep View, which are located in the substrate structure domain. We showed the spatial distribution of Ser30,Val31,Thr54,Ser57,Ser107,Ala108,Thr110,Ala141 amino acid residues in the protein OsHAL3 by Deep View, which contribute to the formation of the hydrogen bonds between OsHAL3 protein and FMN cofactor.To sum up, our results show that Ser28,Lys35, Phe60, Pro105, Ile126, Ala129, Pro140, Leu156, Pro169, Val184, Pro187 are key sites of the PPCDC activity;Glu9, Ser65, Ala98, Pro135,Gly163 sites have few effects on the PPCDC activity of the OsHAL3 protein.