Mutation Analysis of Violaxanthin De-epoxidase Identifies Substrate-binding Sites and Residues Involved in Catalysis

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.In natural environments, light intensity is variable and often exceeds the saturation limit of photosynthesis (1, 2). As a consequence, excitation energy in excess may lead to production of reactive oxygen species and to oxidative stress, in a process called photoinhibition (2, 3). Photosynthetic organisms have evolved several mechanisms to dissipate excess energy safely and to increase the capacity for scavenging reactive oxygen species. A major role is played by the xanthophyll cycle (4, 5) in which the diepoxide xanthophyll violaxanthin is converted into the epoxide-free zeaxanthin. Zeaxanthin is a key molecule for plant photoprotection, being involved in singlet oxygen scavenging as well as singlet chlorophyll quenching (6 -10).Violaxanthin to zeaxanthin conversion is catalyzed by a lumenal enzyme, called violaxanthin de-epoxidase (VDE).3 The reducing power for the reaction is provided by ascorbate (11), probably in its protonated form (12). VDE is activated when light-driven proton translocation across the thylakoid membrane exceeds the dissipation rate of the proton gradient by ATPase, leading to a decrease in pH in the thylakoid lumen. Inactive VDE is a soluble protein, but upon activation, it associates with the thylakoid membrane (13) where its substrate violaxanthin is located (14). When light intensity decreases, the stromal enzyme zeaxanthin epoxidase converts zeaxanthin back to violaxanthin (15, 16). Both 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. Because of their rather low similarity with other lipocalins, their true membership of the lipocalin family has been challenged (17). In additio...

The investigation of violaxanthin de-epoxidation in the primitive green algaMantoniella squamata(Prasinophyceae) indicates mechanistic differences in xanthophyll conversion to higher plants

“…The results obtained for the LHCII and the spinach VDE, i.e. a high ratio of the second de-epoxidation rate (de-epoxidation from Ax to Zx) to the first deepoxidation rate (conversion from Vx to Ax), fit well with the recent model of Vx de-epoxidation (Arnoux et al 2009;Saga et al 2010). This model predicts a dimerization of VDE at low pH-values with the establishment of a channel that is able to incorporate the complete Vx molecule and links the two catalytic sites of the VDE monomers.…”

“…This model predicts a dimerization of VDE at low pH-values with the establishment of a channel that is able to incorporate the complete Vx molecule and links the two catalytic sites of the VDE monomers. According to the structural data presented by Arnoux et al (2009) and Saga et al (2010), the dimeric structure of VDE also contains two putative binding sites for the cosubstrate ascorbate (Asc). The dimer model of VDE proposes that the two catalytic and Asc binding sites allow the de-epoxidation of both epoxy groups of the Vx molecule, thus avoiding the detachment and rebinding of Ax.…”

“…The dimer contains a channel that links the two active sites of the monomers and is able to harbour the complete Vx molecule, thus allowing the de-epoxidation of the two epoxy groups. Investigations employing in silico docking were used to identify putative substrate binding sites and residues involved in catalysis, whose importance was then accessed by mutational analysis (Saga et al 2010).…”

Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity

“…5). For example, the biosynthesis of the carotenoids antheraxanthin and violaxanthin through the xanthophyll cycle is known to be activated under excessive light conditions, as part of a photo-protection mechanism (28), and accordingly showed higher representation in the "light" tissues and cell cultures. Our models suggests that the methylerythritol 4-phosphate (MEP) pathway, which generates the precursors for multiple isoprenoids including the carotenoids (29), is controlled differently than the downstream carotenoid biosynthesis because it is showing constant representation in all tissues and a higher representation in the dark culture model.…”

Characterisation of Carotenoids Involved in the Xanthophyll Cycle

“…Di-epoxy Viola is de-epoxidised to epoxy-free Zea in two-step reaction catalysed by Viola de-epoxidase (VDE) with mono-epoxy Anth as an intermediate product. In absence of photosynthesis, VDE is localised in the thylakoid lumen as inactive monomer; however, it undergoes dimerization and binds to the membrane in an acidic pH caused by the light-driven transmembrane proton gradient [6][7][8]. Additionally, ascorbate as a donor of protons and monogalactosyldiacylglycerol (MGDG) as lipid-forming inverted hexagonal structures are essential for Viola de-epoxidation [9,10].…”