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

Hallin, Erik LU ; Guo, Kuo LU and Åkerlund, Hans-Erik LU (2015) In Photosynthesis Research 124(2). p.191-198
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Photosynthesis Research
volume
124
issue
2
pages
191 - 198
publisher
Springer
external identifiers
  • pmid:25764016
  • wos:000352715500006
  • scopus:84939988612
ISSN
0166-8595
DOI
10.1007/s11120-015-0118-9
language
English
LU publication?
yes
id
f2ff8d24-36c8-4afa-942a-035e8d5709d5 (old id 5261741)
date added to LUP
2015-04-16 14:01:09
date last changed
2017-11-05 03:12:55
@article{f2ff8d24-36c8-4afa-942a-035e8d5709d5,
  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.},
  author       = {Hallin, Erik and Guo, Kuo and Åkerlund, Hans-Erik},
  issn         = {0166-8595},
  language     = {eng},
  number       = {2},
  pages        = {191--198},
  publisher    = {Springer},
  series       = {Photosynthesis Research},
  title        = {Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.},
  url          = {http://dx.doi.org/10.1007/s11120-015-0118-9},
  volume       = {124},
  year         = {2015},
}