Utilization of Dioxygen by Carotenoid Cleavage Oxygenases

Carotenoid cleavage oxygenases (CCOs) are non-heme, Fe(II)-dependent enzymes that participate in biologically important metabolic pathways involving carotenoids and apocarotenoids, including retinoids, stilbenes, and related compounds. CCOs typically catalyze the cleavage of non-aromatic double bonds by dioxygen (O₂) to form aldehyde or ketone products. Expressed only in vertebrates, the RPE65 sub-group of CCOs catalyzes a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters. It remains unclear whether the former group of CCOs functions as mono- or di-oxygenases. Additionally, a potential role for O₂ in catalysis by the RPE65 group of CCOs has not been evaluated to date. Here, we investigated the pattern of oxygen incorporation into apocarotenoid products of Synechocystis apocarotenoid oxygenase. Reactions performed in the presence of ¹⁸O-labeled water and ¹⁸O₂ revealed an unambiguous dioxygenase pattern of O₂ incorporation into the reaction products. Substitution of Ala for Thr at position 136 of apocarotenoid oxygenase, a site predicted to govern the mono- versus dioxygenase tendency of CCOs, greatly reduced enzymatic activity without altering the dioxygenase labeling pattern. Reevaluation of the oxygen-labeling pattern of the resveratrol-cleaving CCO, NOV2, previously reported to be a monooxygenase, using a purified enzyme sample revealed that it too is a dioxygenase. We also demonstrated that bovine RPE65 is not dependent on O₂ for its cleavage/isomerase activity. In conjunction with prior research, the results of this study resolve key issues regarding the utilization of O₂ by CCOs and indicate that dioxygenase activity is a feature common among double bond-cleaving CCOs.     

Identification of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120 with different cleavage activities

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes that oxidatively cleave carotenoids into apocarotenoids. Dioxygenases have been identified in plants and animals and produce a wide variety of cleavage products. Despite what is known about apocarotenoids in higher organisms, very little is known about apocarotenoids and CCDs in microorganisms. This study surveyed cleavage activities of ten putative carotenoid cleavage dioxygenases from five different cyanobacteria in recombinant Escherichia coli cells producing different carotenoid substrates. Three CCD homologs identified in Nostoc sp. PCC 7120 were purified, and their cleavage activities were investigated. Two of the three enzymes showed cleavage of beta,beta-carotene at the 9,10 and 15,15' positions, respectively. The third enzyme did not cleave full-length carotenoids but cleaved the apocarotenoid beta-apo-8'-carotenal at the 9,10 position. 9,10-Apocarotenoid cleavage specificity has previously not been described. The diversity of carotenoid cleavage activities identified in one cyanobacteria suggests that CCDs not only facilitate the degradation of photosynthetic pigments but generate apocarotenals with yet to be determined biological roles in microorganisms.     

The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').     

Modulation of hepatocyte nuclear factor-4alpha function by the peroxisome-proliferator-activated receptor-gamma co-activator-1alpha in the acute-phase response

Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10'-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.     

Identification and characterization of a unique cysteine residue proximal to the catalytic site of Arabidopsis thaliana carotenoid cleavage enzyme 1

AtCCD1 and AtNCED3 are related carotenoid cleavage enzymes from Arabidopsis thaliana that catalyze the oxidative cleavage of, respectively, the 9,10 (9',10') double bonds of carotenoid substrates such as beta-carotene, and the 11,12 double bond of 9-cis epoxycarotenoids. Although the cellular and cleavage functionalities of these enzymes have been reported, their mechanisms and related structural environments mediating these disparate specificities in homologous enzymes have not been well characterized. By relating the differences observed in UV and visible light absorption and Cu(II) electron paramagnetic signals to variations in sequence alignments and 3-D homology models of the two A. thaliana enzymes, we identified a putatively proximal cysteine residue (Cys352) in AtCCD1 that is not conserved in AtNCED3. Spectral analysis of the Cys to Ala mutant confirmed its uniqueness and proximity to the metal binding site, but precluded any role for the residue in the mediation of the observed metal binding affinity or associated steric constraint differences. Further analysis of kinetic substrate cleavage properties indicated a decrease in Vmax and a subtle increase in Km for the C352A mutant compared with those observed for the wild-type, thus confirming catalytic site proximity and suggesting possible roles for the unique cysteine in the modulation of substrate affinity and (or) the reaction rate of AtCCD1.     

Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin.     

Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway

Torulene, a C₄₀ carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C₃₅ xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C₃₅ apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.     

4‐Oxo‐β‐apo‐13‐carotenone from the Cyanobacterium Anabaena cylindrica PCC 7122

Apocarotenoids are widely distributed among living organisms (bacteria, fungi, algae, plants and even animals) and have been associated with several signaling functions. These compounds are generated by the activity of carotenoid cleavage dioxygenases (CCDs), whose diversity greatly contributes to the large number of apocarotenoids that have been described so far. It is nevertheless expected that a considerable diversity of these molecules is yet to be discovered. In this work, we describe the isolation and structural elucidation of the apocarotenoid 4‐oxo‐β‐apo‐13‐carotenone from the cultured freshwater cyanobacterium Anabaena cylindrica PCC 7122, corresponding to the first report of this compound from natural sources.     

Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family

Arabidopsis thaliana has nine genes that constitute a family of putative carotenoid cleavage dioxygenases (CCDs). While five members of the family are believed to be involved in synthesis of the phytohormone abscisic acid, the functions of the other four enzymes are less clear. Recently two of the enzymes, CCD7/MAX3 and CCD8/MAX4, have been implicated in synthesis of a novel apocarotenoid hormone that controls lateral shoot growth. Here, we report on the molecular and genetic interactions between CCD1, CCD7/MAX3 and CCD8/MAX4. CCD1 distinguishes itself from other reported CCDs as being the only member not targeted to the plastid. Unlike ccd7/max3 and ccd8/max4, both characterized as having highly branched phenotypes, ccd1 loss-of-function mutants are indistinguishable from wild-type plants. Thus, even though CCD1 has similar enzymatic activity to CCD7/MAX3, it does not have a role in synthesis of the lateral shoot growth inhibitor. Rather, it may have a role in synthesis of apocarotenoid flavor and aroma volatiles, especially in maturing seeds where loss of function leads to significantly higher carotenoid levels.     

Enzymology of vertebrate carotenoid oxygenases

Mammals and higher vertebrates including humans have only three members of the carotenoid cleavage dioxygenase family of enzymes. This review focuses on the two that function as carotenoid oxygenases. β-Carotene 15,15′-dioxygenase (BCO1) catalyzes the oxidative cleavage of the central 15,15′ carbon-carbon double of β-carotene bond by addition of molecular oxygen. The product of the reaction is retinaldehyde (retinal or β-apo-15-carotenal). Thus, BCO1 is the enzyme responsible for the conversion of provitamin A carotenoids to vitamin A. It also cleaves the 15,15′ bond of β-apocarotenals to yield retinal and of lycopene to yield apo-15-lycopenal. β-Carotene 9′,10′-dioxygenase (BCO2) catalyzes the cleavage of the 9,10 and 9′,10′ double bonds of a wider variety of carotenoids, including both provitamin A and non-provitamin A carotenoids, as well as the xanthophylls, lutein and zeaxanthin. Indeed, the enzyme shows a marked preference for utilization of these xanthophylls and other substrates with hydroxylated terminal rings. Studies of the phenotypes of BCO1 null, BCO2 null, and BCO1/2 double knockout mice and of humans with polymorphisms in the enzymes, has clarified the role of these enzymes in whole body carotenoid and vitamin A homeostasis. These studies also demonstrate the relationship between enzyme expression and whole body lipid and energy metabolism and oxidative stress.In addition, relationships between BCO1 and BCO2 and the development or risk of metabolic diseases, eye diseases and cancer have been observed. While the precise roles of the enzymes in the pathophysiology of most of these diseases is not presently clear, these gaps in knowledge provide fertile ground for rigorous future investigations.This article is part of a Special Issue entitled Carotenoids: Recent Advances in Cell and Molecular Biology edited by Johannes von Lintig and Loredana Quadro.     

Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon

Carotenoids are nutritionally important tetraterpenoid pigments that upon oxidative cleavage give rise to apocarotenoid (norisoprene) aroma volatiles. beta-Carotene is the predominant pigment in orange-fleshed melon (Cucumis melo L.) varieties, reaching levels of up to 50 microg/gFW. Pale green and white cultivars have much lower levels (0-10 microg/gFW). In parallel, beta-ionone, the 9,10 cleavage product of beta-carotene, is present (12-33ng/gFW) in orange-fleshed melon varieties that accumulate beta-carotene, and in much lower levels (0-5 ng/gFW) in pale green and white fleshed varieties. A search for a gene putatively responsible for the cleavage of beta-carotene into beta-ionone was carried out in annotated melon fruit EST databases yielding a sequence (CmCCD1) highly similar (84%) to other plant carotenoid cleavage dioxygenase genes. To test its function, the clone was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. We show here that the CmCCD1 gene product cleaves carotenoids at positions 9,10 and 9',10', generating geranylacetone from phytoene; pseudoionone from lycopene; beta-ionone from beta-carotene, as well as alpha-ionone and pseudoionone from delta-carotene. CmCCD1 gene expression is upregulated upon fruit development both in orange, pale-green and white melon varieties, despite the lack of apocarotenoid volatiles in the later. Thus, the accumulation of beta-ionone in melon fruit is probably limited by the availability of carotenoid substrate.     

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).