Rc. Bugos et Hy. Yamamoto, MOLECULAR-CLONING OF VIOLAXANTHIN DE-EPOXIDASE FROM ROMAINE LETTUCE AND EXPRESSION IN ESCHERICHIA-COLI, Proceedings of the National Academy of Sciences of the United Statesof America, 93(13), 1996, pp. 6320-6325
Plants need to avoid or dissipate excess light energy to protect photo
system II (PSII) from photoinhibitory damage. Higher plants have a con
served system that dissipates excess energy as heat in the light-harve
sting complexes of PSII that depends on the transthylakoid Delta pH an
d violaxanthin de-epoxidase (VDE) activity, To our knowledge, we repor
t the first cloning of a cDNA encoding VDE and expression of functiona
l enzyme in Escherichia coli, VDE is nuclear encoded and has a transit
peptide with characteristic features of other lumen-localized protein
s. 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 cy
steine-rich region, (ii) a lipocalin signature, and (iii) a highly cha
rged region, The E. coil expressed enzyme de-epoxidizes violaxanthin s
equentially to antheraxanthin and zeaxanthin, and is inhibited by dith
iothreitol, similar to VDE purified from chloroplasts. This confirms t
hat the cDNA encodes an authentic VDE of a higher plant and is unequiv
ocal evidence that the same enzyme catalyzes the two-step mono de-epox
idation reaction, The cloning of VDE opens new opportunities for exami
ning the function and evolution of the xanthophyll cycle, and possibly
enhancing tight-stress tolerance of plants.