The Arabidopsis LUT1 locus encodes a member of the cytochrome P450 family that is required for carotenoid ε-ring hydroxylation activity

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Abstract
Lutein, a dihydroxy xanthophyll, is the most abundant carotenoid in plant photosynthetic tissues and plays crucial structural and functional roles in the light-harvesting complexes. Carotenoid β-and ε-hydroxylases catalyze the formation of lutein from α-carotene (β,ε-carotene). In contrast to the well studied β-hydroxylases that have been cloned and characterized from many organisms, the ε-hydroxylase has only been genetically defined by the lut1 mutation in Arabidopsis . We have isolated the LUT1 gene by positional cloning and found that, in contrast to all known carotenoid hydroxylases, which are the nonheme diiron monooxygenases, LUT1 encodes a cytochrome P450-type monooxygenase, CYP97C1. Introduction of a null mutant allele of LUT1, lut1 - 3 , into the β-hydroxylase 1/β-hydroxylase 2 ( b1 b2 ) double-mutant background, in which both Arabidopsis β-hydroxylases are disrupted, yielded a genotype ( lut1 - 3 b1 b2 ) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated β-rings were still produced in lut1 - 3 b1 b2 , suggesting that a fourth unknown carotenoid β-hydroxylase exists in vivo that is structurally unrelated to β-hydroxylase 1 or 2. A second chloroplast-targeted member of the CYP97 family, CYP97A3, is 49% identical to LUT1 and hypothesized as a likely candidate for this additional β-ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron β-hydroxylases.