Abstract
Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.
Highlights
The stromal enzyme zeaxanthin epoxidase converts zeaxanthin back to violaxanthin [15, 16]. Both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase have been suggested to belong to lipocalins, a multigenic protein family characterized by a conserved structural organization with an 8-strand -barrel [15]
VDE and zeaxanthin epoxidase are classified among outlier lipocalins because they do not present all three conserved regions typical of this multigenic family
Docking of Violaxanthin in VDE putative lipocalin domain (VDEcd) Structure at pH 5—After VDEcd crystals at pH 5 had been obtained, one main objective was to reveal the structure of the enzyme-substrate complex
Summary
Docking—The ligands, violaxanthin and ascorbic acid, were docked into the crystal structure of the active form of the VDE lipocalin domain (VDEcd) obtained at pH 5 (Protein Data Bank code 3CQR [22]), using AUTODOCK 4.0 [23]. The minimal distances between violaxanthin, ascorbate, and the protein were calculated by means of p3d [27] and PyMOL [48]. The latter was used to generate all images. 40 l of partially purified VDE was added to 120 g of chlorophyll of thylakoids and 40 l of buffers with variable pH values (MES, HEPES, or Tris). The presence of VDE in supernatants (and pellets) was accessed by an antibody against the His tag, which binds the recombinant protein
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