title: 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.
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, P.O. Box 124, 221 00, Lund, Sweden.
authors.forename: Erik Ingmar
authors.initials: EI
authors.lastname: Hallin
authors.forename: Kuo
authors.initials: K
authors.lastname: Guo
authors.forename: Hans-Erik
authors.initials: HE
authors.lastname: Åkerlund
mesh_terms: D000595:Amino Acid Sequence; D003553:Cystine; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D018724:Spinacia oleracea / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D020524:Thylakoids / Q000201:enzymology; D024341:Xanthophylls / Q000378:metabolism; D065146:Zeaxanthins / Q000378:metabolism*
chemical_list: D004220:Disulfides; D024341:Xanthophylls; D065146:Zeaxanthins; D003553:Cystine; C005613:violaxanthin; D010088:Oxidoreductases; C023290:violaxanthin de-epoxidase