Acid Phosphatase Activity And Preliminary Crystallographic Analysis Of VSP1 From Arabidopsis Thaliana

Vegetative storage proteins (VSP) are known for the temporary storage of nitrogen, which have been identified from numerous plants. They can accumulate to a high abundance in various storage organs. As temporary storage of amino acids, they are degraded when plants need nitrogen and other nutrients. VSPs were identified from soybean leaves at the earliest, and to distinguish them from the well-characterized seed storage proteins, we have called them vegetative storage proteins.Arabidopsis thaliana VSP1 and VSP2 are homologous proteins of soybean VSP. They are approximately 40% identical to the soybean VSP sequence. Arabidopsis VSPs mainly accumulate in flowers and buds. Arabidopsis VSPs transcripts can be induced by mechanical wound, jasmonic acid (JA), insect herbivory, phosphate deprivation, osmotic and nutritional stresses. Compared with soybean VSP, Arabidopsis VSPs have obvious acid phosphatase activity and are classified as haloacid dehalogenase (HAD) superfamily. Recombinant Arabidopsis VSP2 is an anti-insect protein and this activity is correlated with its acid phosphatase acitivity.However, the structure of Arabidopsis VSPs hasn't been reported, impeding people understanding its catalytic mechanism. We carried out enzymatic and crystall-ographic studies of recombinant VSP1, which laid the foundation for resolving VSP1 structure and clarifying its catalytic mechanism.Arabidopsis VSP1-pET-22b plasmid was transformed into E. coli BL21(DE3) and VSP1 was highly expressed in E. coli BL21(DE3) cells. Recombinant VSP1 was purified via one-step Ni-NTA affinity chromatography. The studies on acid phosphatase activity of Arabidopsis VSP1 contained catalysis difference of divalent copper ion and magnesium ion, substrate specificity, and inhibitor effects. The effects of metal ions concentrations were different between Cu2+ and Mg2+. When concentration of Cu2+ was 1 mM, VSP1 activity was the highest, and then decreased; when concentration of Mg2+ was 10 mM, VSP1 had the highest activity, and then essentially unchanged. These suggested two divalent metal ions had different activation mechanism. The recombinant VSP1 displayed narrow substrate specificity. The aryl phosphates pNPP and MUP were the most readiy hydrolyzed substrates. Most other substrates tested were hydrolyzed at less than 5% the rate of pNPP. Inorganic phosphate had little effect on phosphatase activity. EDTA, molybdate and vanadate had significant inhibitory effect. VSP1 mutants on Asp-124 residue were constructed and the mutants had no acid phosphatase activity. This result suggested that Asp-124 was necessary for the catalytic function of VSP1.Crystallizations were carried out at 287 K using the sitting-drop vapor diffusion method and the initial trials were performed using Crystal Screen kitsⅠ&Ⅱfrom Hampton Research and WizardⅠ&Ⅱfrom Emerald BioSystems. We found VSP1 crystals in No.40 in Crystal Sreen I. The crystal diffracted to 1.9 A resolution and a complete X-ray data set was collected at 100 K using Cu Ka radiation from a rotating-anode X-ray source. Preliminary data shows VSP1 crystals belonged to the space group C2 with unit cell parameters a=123.1A, b=48.4A, c=85.6A,α=γ= 90,β= 116.3°.