Key Amino Acids In Redox Regulation And The Function Of Stress Tolerance In Plant VKORs

Chloroplast is an important organelle in photosynthesis. Many proteins in chloroplast are regulated by redox through the disulfide bond/hydrosulphonyl transformation to realize its activity. Plant thylakoid membrane protein Vitamin K epoxide reductase (VKOR) is found to catalyze the disulfide bond formation. It is also known as Lumen Thiol Oxidoreductase1(LTO1). Research of the function of VKOR and its essential amino acid residues has important theoretical significance to clarify the chloroplast redox regulation mechanism. This research constructed procaryotic and eukaryotic expression vector of Arabidopsis VKOR (AtVKOR) and detected the activity in vivo and in vitro. The essential amino acids were forecasted using molecular docking. Site-directed mutagenesis was used to study the AtVKOR combination with the substrate and important amino acids in catalyzing the process of electron transfer. In addition, we found that the Solanum lycopersicum VKOR (SIVKOR) has osmotic stress resistance function. The results are as follows:(1) We built the AtVKOR eukaryotic expression system and detected the phylloquinone reduction activity. Early research has shown that prokaryotic system and Pichia pastoris expression system has certain defects to express the membrane protein AtVKOR. This study built Saccharomyces cerevisiae eukaryotic expression system using galactose as inducer. In the presence of the substrate phylloquinone and the reductant DTT,AtVKOR reduce phylloquinone to hydroquinone. We used high pressure liquid chromatography (HPLC) to detect the conversion of phylloquinone and hydroquinone and quantifed the AtVKOR activity.(2) Several amino acids of AtVKOR were identified to involve in the binding of phylloquinone by molecular docking. Through homologous modeling, three-dimensional structure of AtVKOR was established using Synechococcus sp. VKOR crystal structure as a template. Then we obtained the molecular docking model using substrate phylloquinone as the ligand. Docking results showed that most important amino acids of AtVKOR combining quinone were hydrophobic. According to the result of plant VKORs homology comparison, the conservative amino acids in all plant VKORs (Ser77, Leu87, Phe137and Leu141) adjacent to the naphthoquinone ring may play an important role in combination with the substrate phylloquinone and catalyzing enzyme activity.(3) Amino acid residues binding phylloquinone directly involved in the activity of phylloquinone reduction. According to the result of molecular docking, we got AtVKOR mutant protein. Mutants, Ser77Ala and Phe137Ala, lost the activity of phylloquinone reduction totally; Leu87Ala and Leu141Ala mutants decreased significantly, only27.88%and41.46%as that of wild-type respectively while Leu141Arg lost activity completely.(4) The residues above binding phylloquinone were also involved in the disulfide bond formation of substrate protein. AtVKOR catalyzed a series of electron transfer between substrate protein and phylloquinone. In vivo, motility complementation assay and β-galactosidase activity assay were used to detect these amino acids in catalyzing the formation of disulfide bonds. The results showed that all the mutant oxidation ability were decreased, especially Ser77Ala and Leu141Arg mutants, only39.68%and46.03%as that of wild-type respectively. This is consistent with the result of reduction activity decline.(5) The conservative cysteines in VKOR domain played critical role in the activity of phylloquinone reduction. When each of the four conservative cysteines was mutated to alanine respectively, all mutants lost activity entirely. It suggested that the four conservative cysteines were involved in electron transfer of oxidoreduction both through the reduction of phylloquinone and the oxidation of Trx-like domain.(6) SlVKOR has certain resistance to salt stress and drought stress. The recombinant SlVKOR expressed in Escherichia coli enhanced the tolerance to salt stress. We obtained sense and antisense SIVKOR transgenic tomato homozygotes at T2generation. Over-expression lines showed a lower level of ROS compared with that of WT plants after salt or drought treatment. The activities of reactive oxygen scanvener enzymes, including SOD, POD, APX, and CAT were consistent with the accumulation of ROS. According to the results, we suggested that SlVKOR was involved in resistance to salt or drought stress.