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 -and -hydroxylases catalyze the formation of lutein from ␣-carotene (,-carotene). In contrast to the well studied -hydroxylases that have been cloned and characterized from many organisms, the -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 -hydroxylase 1͞-hydroxylase 2 (b1 b2) double-mutant background, in which both Arabidopsis -hydroxylases are disrupted, yielded a genotype (lut1-3 b1 b2) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated -rings were still produced in lut1-3 b1 b2, suggesting that a fourth unknown carotenoid -hydroxylase exists in vivo that is structurally unrelated to -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 -ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron -hydroxylases. Carotenoids are terpenoid compounds that perform a variety of critical roles in photosystem structure, light harvesting, and photoprotection. Lutein (3R,3ЈR-,-carotene-3,3Ј-diol), is the most abundant carotenoid in all plant photosynthetic tissues, in which it plays an important role in light-harvesting complex II assembly and function. Zeaxanthin (3R,3ЈR-,-carotene-3,3Ј-diol) is a structural isomer of lutein and is a critical component of nonphotochemical quenching (1, 2). The synthesis of lutein and zeaxanthin involves cyclization of lycopene to form ␣-and -carotene, respectively, followed by the introduction of hydroxyl groups onto the ionone rings by a class of enzymes known as carotenoid hydroxylases (Fig. 1). -Hydroxylases add hydroxyl groups to carbon 3 (C-3) of -rings, whereas hydroxylation of C-3 on -rings is carried out by -hydroxylases. Two -ring hydroxylations of -carotene yield zeaxanthin, whereas one -ring and one -ring hydroxylation of ␣-carotene yield lutein (Fig. 1).Based on the stereospecific introduction of C-3 hydroxyl groups and the requirement for molecular oxygen, carotenoid hydroxylation reactions were predicted to be catalyzed by mixedfunction oxygenases such as the cytochrome P450 enzymes (3-5). However, -hydroxylases have been cloned from a variety of photosynthetic and nonphotosynthetic bacteria, green algae, and plants (6) and in all three phyla encode nonheme diiron proteins that have a fundamentally different hydroxylation reaction mechanism than heme-binding cytochro...
Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of  -and -ring carotenoid hydroxylases. Two genes that encode  -ring hydroxylases (  -hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward  -rings but only weakly active toward -rings. A third distinct activity required for -ring hydroxylation has been defined by mutation of the LUTEIN1 ( LUT1 ) locus, but LUT1has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to  -hydroxylases 1 and 2 ( b1 and b2 , respectively) were isolated and all possible hydroxylase mutant combinations were generated.  -Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known  -hydroxylase enzymes, still contains significant levels of  -carotene-derived xanthophylls, suggesting that additional  -ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of  -carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated  -rings. This finding suggests that it is the presence of minimal amounts of hydroxylated  -rings, rather than minimal amounts of specific  -carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.
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