Document Detail

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.
MedLine Citation:
PMID:  8692813     Owner:  NLM     Status:  MEDLINE    
Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.
R C Bugos; H Y Yamamoto
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  93     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1996 Jun 
Date Detail:
Created Date:  1996-08-23     Completed Date:  1996-08-23     Revised Date:  2010-09-13    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  6320-5     Citation Subset:  IM    
Department of Plant Molecular Physiology, University of Hawaii, Honolulu 96822, USA.
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MeSH Terms
Amino Acid Sequence
Base Sequence
Chromatography, Affinity
Cloning, Molecular
DNA, Complementary
Electrophoresis, Polyacrylamide Gel
Escherichia coli / genetics
Lettuce / enzymology*
Molecular Sequence Data
Molecular Weight
Oxidoreductases / chemistry,  genetics*,  isolation & purification
Reg. No./Substance:
0/DNA, Complementary; EC 1.-/Oxidoreductases; EC 1.-/violaxanthin de-epoxidase

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