Overexpression of violaxanthin de-epoxidase: properties of C-terminal deletions on activity and pH-dependent lipid binding.
Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A full-length Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the specific activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity. The difficulties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.
Hieber
A David
AD
Department of Molecular Biosciences, University of Hawaii-Manoa, Honolulu 96822, USA.
Bugos
Robert C
RC
Verhoeven
Amy S
AS
Yamamoto
Harry Y
HY
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008027:Light; D050356:Lipid Metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D017384:Sequence Deletion; D020524:Thylakoids / Q000201:enzymology; D014026:Nicotiana / Q000235:genetics; D024341:Xanthophylls / Q000378:metabolism; D065146:Zeaxanthins; D019207:beta Carotene / Q000031:analogs & derivatives* / Q000378:metabolism
D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene; C031140:antheraxanthin; C005613:violaxanthin; D010088:Oxidoreductases; C023290:violaxanthin de-epoxidase