Molecular studies on structural changes and oligomerisation of violaxanthin de-epoxidase associated with the pH-dependent activation

Abstract

Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.

A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health

“…Carotenoid biosynthetic enzymes appear to be either associated to envelope and thylakoid membranes (PSY, PDS, ZDS, CRTISO, LCYB, LCYE, CYP97A and CYP97C) or integral membrane proteins (ZISO, BCH) [43,63]. VDE attaches to the thylakoid membrane at acidic pH but behaves as a soluble lumenal protein at neutral pH [89]. PSY has also been found in plastoglobules or the stroma of chloroplasts from different species [64,90], while in the plastids of dark-germinated seedlings (i.e.…”

Lipid Dependence of Xanthophyll Cycling in Higher Plants and Algae

“…Activation of VDE is probably driven by a dimerization of the water-soluble monomeric VDE, followed by the binding of the active, dimeric VDE to the lumen side of the thylakoid membrane (Hager and Holocher, 1994;Arnoux et al, 2009;Saga et al, 2010). With regard to the dimerization it has been proposed that the C-terminus of the VDE plays a role in the interaction of the VDE monomers (Hallin et al, 2016) and that four specific amino acid residues are important for the pH-dependent activation (Fufezan et al, 2012). In addition, it has been suggested that the conserved cysteine residues and the disulfide bridges formed by the cysteines are sensitive to redox changes of the thylakoid membrane induced by the photosynthetic electron transport (Hallin et al, 2015;Simionato et al, 2015).…”

Overexpression of the ChVDE gene, encoding a violaxanthin de-epoxidase, improves tolerance to drought and salt stress in transgenic Arabidopsis

“…VDE is the first described putative plant lipocalin (Bugos and Yamamoto 1996) and has been classified as a lipocalinlike protein (Charron et al 2005). The activation of VDE is related to the conformational change (Kawano and Kuwabara 2000) and the formation of an ɑ-helical coiled structure located at the C-terminal domain (Hallin et al 2016). Other research has reported that VDE uses ascorbic acid as a secondary substrate, possibly as a monomer.…”