Defining the primary route for lutein synthesis in plants: The role of Arabidopsis carotenoid -ring hydroxylase CYP97A3

Lutein, a dihydroxy derivative of -carotene ( , -carotene), is the most abundant carotenoid in photosynthetic plant tissues where it plays important roles in light-harvesting complex-II structure and function. The synthesis of lutein from lycopene requires at least four distinct enzymatic reactions: and -ring cyclizations and hydroxylation of each ring at the C-3 position. Three carotenoid hydroxylases have already been identified in Arabidopsis, two nonheme diiron -ring monooxygenases (the B1 and B2 loci) that primarily catalyze hydroxylation of the -ring of , -carotenoids and one heme-containing monooxygenase (CYP97C1, the LUT1 locus) that catalyzes hydroxylation of the -ring of , -carotenoids. In this study, we demonstrate that Arabidopsis CYP97A3 (the LUT5 locus) encodes a fourth carotenoid hydroxylase with major in vivo activity toward the -ring of -carotene ( , -carotene) and minor activity on the -rings of -carotene ( , -carotene). A cyp97a3-null allele, lut5-1, causes an accumulation of -carotene at a level equivalent to -carotene in wild type, which is stably incorporated into photosystems, and a 35% reduction in -carotene-derived xanthophylls. That lut5-1 still produces 80% of wild-type lutein levels, indicating at least one of the other carotene hydroxylases, can partially compensate for the loss of CYP97A3 activity. From these data, we propose a model for the preferred pathway for lutein synthesis in plants: ring cyclizations to form -carotene, -ring hydroxylation of -carotene by CYP97A3 to produce zeinoxanthin, followed by -ring hydroxylation of zeinoxanthin by CYP97C1 to produce lutein.