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

Abstract

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.␣-carotene ͉ carotenoid hydroxylase ͉ cytochrome P450 ͉ xanthophyl ͉ substrate channeling C arotenoids are a group of Ͼ600 red, yellow, and orange pigments (most commonly C 40 ) that contain extended conjugated double-bond systems (1, 2). This tremendous structural diversity has presumably evolved in relation to the many functions of carotenoids, which include acting as structural components of membranes and photosystems, accessory light-harvesting pigments, components for photoprotection and substrates for hormone syntheses (3, 4). Carotenoids are derived from isoprenoid precursors and are generally divided into two groups, the carotenes (acyclic or cyclic hydrocarbons) and the xanthophylls (oxygenated derivatives of carotenes). The carotenes of bacteria, plants, and protists are chemically and structurally similar, indicating that the initial steps of carotene synthesis are similar among organisms (3). Much of the enormous structural diversity of carotenoids occurs in the later steps of synthesis, including various ring cyclization and oxygenation reactions.Cyclization of lycopene is a key step in generating carotenoid diversity because it marks a branch point to two major cyclic carotenoid groups ( Fig. 1): the ␤,␤-and ␤,-carotenoids. ␤,␤-Carotenoids contain two identical ␤-rings formed by the symmetrical action of the ␤-ring cyclase (␤-cyclase), whereas ␤,-carotenoids contain two different ring stru...

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

“…The second possibility was that perhaps the CYP97 enzymes did not function optimally as individual enzymes but required coexpression and interaction, which would allow for efficient hydroxylation of a mixed-ring compound, such as a-carotene. Biochemical phenotypes of plant knockouts support the hypothesis that CYP97 enzymes act sequentially (first CYP97A and then CYP97C) to hydroxylate a-carotene (Kim and DellaPenna, 2006).…”

“…Although further research is needed to understand the connection between interaction and the efficiency of dihydroxylation in planta, we hypothesize that the ability to form a protein complex improves the efficiency of hydroxylation of dualringed carotene substrates. The hypothesis based on plant mutants is that in carotenoid biosynthesis, CYP97A functions first to produce the monohydroxylated carotene, zeinoxanthin, which is transferred by some unknown mechanism as the substrate for CYP97C (Kim and DellaPenna, 2006;Kim et al, 2009). In those studies, CYP97C mutants accumulated substantially higher levels of the monohydroxylated carotene, compared with CYP97A mutants, although the levels were not what would be expected on the basis of wild-type levels of lutein formed in leaf tissue.…”

“…From genetic and functional complementation studies Kim and DellaPenna, 2006;Quinlan et al, 2007), it is generally accepted that CYP97C is an «-ring hydroxylase and CYP97A is a b-ring hydroxylase. Mutant phenotypes suggested that CYP97A hydroxylates the b-rings of a-carotene to form zeinoxanthin, which is the preferred substrate for «-ring hydroxylation by CYP97C (Kim and DellaPenna, 2006).…”

“…The pathway likely functions as a multienzyme complex(es) to facilitate metabolite channeling, as predicted by the absence of pathway intermediates and the presence of complexes containing carotenoid biosynthetic enzymes (Maudinas et al, 1977;Camara et al, 1982;Kreuz et al, 1982;AlBabili et al, 1996;Bonk et al, 1997;Lopez et al, 2008). In this study, we examined an intriguing portion of the pathway, where hydroxylation of rings in carotenes is catalyzed by two structurally distinct enzymes, P450 heme (CYP97) and nonheme diiron (HYD) enzymes DellaPenna, 2001, 2004;Kim and DellaPenna, 2006;Quinlan et al, 2007). Hydroxylation of the two b-ionone rings in b-carotene leads to a formation of zeaxanthin, while hydroxylation of the one b-ring and one «-ring in a-carotene leads to lutein.…”

Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii

“…On the other hand, the CYP97C3 and CYP97A5 hydroxylases found in Chlamydomonas have high similarity with the corresponding "-and -hydroxylases of Arabidopsis, CYP97C1 and CYP97A3, respectively (Tian et al, 2004;Kim & DellaPenna, 2006), with identities/similarities of 56%/72% for the CYP97As and 61%/74% for the CYP97Cs (Lohr et al, 2005;Lohr, 2009). In Arabidopsis, the function of these two unrelated hydroxylases families has been well studied using multiple mutants and performing substrate specificity studies by complementation experiments (Kim et al, 2009).…”