Gene And Protein Structure And Function Of A Tau Class Glutathione Transferase(PtGSTU1) From Pinus Tabuliformis

Genetic variation is the prerequisite for forest trees to adapt to complex and changing environments.Understanding the genetic variation in forest tree populations and their fnctional significance is important for tree breeding as it assists more effective targeted breeding program.Gene mutation is an important type of heritable variation.A deep understanding of the relationship between gene mutation and protein structure and function is the premise of effective utilization of genetic variation and the basis of molecular breeding.In this study,Pt GSTU1,a Tau class glutathione transferase from Pinus tabuliformis was targeted to investigate the effect of amino acid site substitutions on the protein structure and biochemical function of the GST protein by three-dimensional structure simulation,site-directed mutation and enzyme function assays.The main results are as follows:1)The sites that can form hydrogen bond between N-terminal domain and C-terminal domain of Pt GSTU1 protein of Pinus tabuliformis were identified.The three-dimensional structure of Pt GSTU1 protein was obtained by homologous modeling with Ta GSTU4 of Aegilops tauschii crystal structure as template.The results show that Pt GSTU1 monomer has a typical three-dimensional structure of GST,with two domains of N-terminal and C-terminal.The N-terminal domain and C-terminal domain are connected by a linker region.We identified four pairs of amino acids between the N-terminal and C-terminal domains of Pt GSTU1 protein monomers that can form intramolecular hydrogen bonds: the 18 th arginine and the 103 rd aspartic acid(R18-D103),the 72 nd glutamate and the 96 th arginine(E72-R96),the 12 th alanine and the 169 th tryptophan(A12-W169),the 84 th phenylalanine and the 158 th tyrosine(F84-Y158).Based on this result,we speculate that these hydrogen bonds can stabilize the structure of protein monomer.2)The effects of these four pairs of amino acid sites on the structure and function of the protein were examined.We use site-directed mutation to mutate these amino acids into alanine Ala,which shortens the polar side chain as much as possible and disrupts the formation of intramolecular hydrogen bond.It was found that the stability of the protein was affected after the four pairs of amino acid sites were mutated,but the change of different amino acid sites had different effects on the substrate activity.The results showed that these four pairs of amino acids are the key amino acids that stabilize the monomer structure.3)The effects of amino acid residues with different polarity and conformation on hydrogen bond formation and protein stability were further explored.Arg18 was mutated into alanine Ala,isoleucine Ile,tryptophan Trp,lysine Lys,glutamate Glu and histidine His,and followed by enzyme catalytic activity and structural stability assays.The results showed that none of the six Arg18 mutants could obtain high-purity soluble protein with the correct folding.The mutation of Asp103,which formed a hydrogen bond with Arg18,would not completely inactivate the protein,but significantly reduce its catalytic ability and stability.This means that the mutation of Arg18 has a greater impact on the protein structure than that of Asp103,which verifies the conservatism of N-terminal and the variability of C-terminal domain of GST protein in plants,and indicated that there might be other amino acid sites in C-terminal domain that could form hydrogen bond with Arg18,thus stabilizing the folding structure of protein monomer.This study combined 3D protein structure simulation,site-directed mutation and enzymatic properties to systematically examine the importance of hydrogen bonds on stabilizing the structure of GST protein monomers,and illustrate the impact of genetic variation on the structure and function of GST protein.