Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability

Abstract

Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9 %. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9–C27, C14–C21, C33–C50, C37–C46, C65–C72 and C118–C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.

Lipid Dependence of Xanthophyll Cycling in Higher Plants and Algae

“…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). Changes of the thylakoid redox potential may play a role in the regulation and activation of the VDE via dithiol/disulfide exchange reactions.…”

Exploring the Valuable Carotenoids for the Large-Scale Production by Marine Microorganisms

“…Violaxanthin de-epoxidation and the violaxanthin cycle were first studied in the late 1960s to the early 1970s [ 113 , 114 , 115 ]. Violaxanthin de-epoxidases are susceptible to DTT [ 116 , 117 ], and their thermostability is due to the disulfide bridges present in their structures [ 118 ]. These interconversion mechanisms have been observed to correlate directly to the dissipation of excess excitation energy in leaves in 2% O 2 , 0% CO 2 [ 119 ].…”

Carotenoids from Haloarchaea and Their Potential in Biotechnology

“…Some of the most important carotenoids in terms of biotechnological and biomedical uses explored so far are: Astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione) [ 36 , 39 , 40 ], β-Carotene (β,β-carotene) [ 38 , 41 , 42 , 43 , 44 ], Canthaxanthin (β,β-carotene-4,4′-dione) [ 45 , 46 , 47 , 48 ], β-Cryptoxanthin (hydroxy-β-carotene) [ 38 , 49 , 50 , 51 , 52 , 53 , 54 ], Fucoxanthin [ 38 , 55 ], Lycopene (ψ,ψ-carotene) [ 33 , 56 , 57 ], Lutein (β,ε-carotene-3,3′-diol) [ 42 , 58 , 59 , 60 , 61 ], Zeaxanthin (β,β-carotene-3,3′-diol) [ 38 , 62 , 63 ], and Violaxanthin (5,6:5′,6′-diepoxy-5,5′,6,6′-tetrahydro-β-carotene-3,3′-diol) [ 64 , 65 , 66 , 67 , 68 ].…”