beta-carotene and beta-apo-10--carotenal

Topics: beta Carotene; Carotenoids; Heterocyclic Compounds, 3-Ring; Lactones; Oryza; Plant Growth Regulators

β-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of β-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. β-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of β-apo-8'-carotenal into cells, and β-apo-8'-carotenal was largely converted to β-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-β-apo-8'-carotenol. There was also rapid uptake of β-apo-10'-carotenal into cells, and β-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-β-apo-10'-carotenol and a minor metabolite that is likely a dihydro-β-apo-10'-carotenol. Finally, there was rapid cellular uptake of β-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary β-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.

Topics: beta Carotene; Caco-2 Cells; Carotenoids; Chromatography, High Pressure Liquid; Humans; Kinetics; Mass Spectrometry; Vitamin A

Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis-configured β-carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used

Topics: beta Carotene; Biocatalysis; Carbon Isotopes; Carotenoids; Dioxygenases; Escherichia coli; Gene Expression; Lactones; Oxygen Isotopes; Pisum sativum; Plant Growth Regulators; Plant Proteins; Recombinant Proteins

A novel β-carotene-9,10'-oxygenase (ScBCO2) has been characterized from Saccharomyces cerevisiae ULI3 to convert β-carotene to β-apo-10'-carotenal, which is a precursor of the plant hormone strigolactone.. The ScBCO2 enzyme was purified to homogeneity by ammonium sulfate precipitation, Q sepharose and Superdex-200 chromatography. The molecular mass of the enzyme was ~50 kDa by SDS-PAGE. The purified ScBCO2 enzyme displayed optimal activity at 45 °C and pH 8. Tween 20 (1%, w/v), Trition X-100 (1%, w/v), Mg(2+) (5 mM), Zn(2+) (5 mM), Cu(2+) (5 mM), Ca(2+) (5 mM) or DTT (5 mM) increased in the activity by 3, 7, 14, 17, 23, 26 and 27%, respectively. ScBCO2 only exhibited cleavage activity towards carotenoid substrates containing two β-ionone rings and its catalytic efficiency (kcat/Km) followed the order β-carotene > α-carotene > lutein.. ScBCO2 could be used as a potential candidate for the enzymatic biotransformation of β-carotene to β-apo-10'-carotenal in biotechnological applications.

Topics: beta Carotene; Carotenoids; Chemical Precipitation; Chromatography, Liquid; Dioxygenases; Enzyme Activators; Humans; Hydrogen-Ion Concentration; Infant, Newborn; Molecular Weight; Oxidation-Reduction; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Temperature

The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.

Topics: Acetates; Arabidopsis; Arabidopsis Proteins; beta Carotene; Biocatalysis; Carotenoids; cis-trans-Isomerases; Dioxygenases; Drug Design; Enzyme Inhibitors; Hydrogen-Ion Concentration; Hydroxamic Acids; Molecular Structure; Oryza; Peptide Fragments; Plant Growth Regulators; Plant Proteins; Recombinant Proteins; Silver Compounds; Stereoisomerism; Substrate Specificity

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15'-oxygenase (BCO1) and the β-carotene-9',10'-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15'-apocarotenoids, C20), whereas eccentric cleavage by BCO2 produces long-chain (>C20) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1(-/-), Bco2(-/-), and Bco1(-/-)Bco2(-/-) double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10'-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.

Topics: Animals; beta Carotene; beta-Carotene 15,15'-Monooxygenase; Carotenoids; Cryptoxanthins; Dioxygenases; Hep G2 Cells; Humans; Mice; Mice, Knockout; Vitamin A; Vitamin A Deficiency; Xanthophylls

Strigolactones, phytohormones with diverse signaling activities, have a common structure consisting of two lactones connected by an enol-ether bridge. Strigolactones derive from carotenoids via a pathway involving the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8) and the iron-binding protein D27. We show that D27 is a β-carotene isomerase that converts all-trans-β-carotene into 9-cis-β-carotene, which is cleaved by CCD7 into a 9-cis-configured aldehyde. CCD8 incorporates three oxygens into 9-cis-β-apo-10'-carotenal and performs molecular rearrangement, linking carotenoids with strigolactones and producing carlactone, a compound with strigolactone-like biological activities. Knowledge of the structure of carlactone will be crucial for understanding the biology of strigolactones and may have applications in combating parasitic weeds.

Topics: Arabidopsis; Arabidopsis Proteins; beta Carotene; Biosynthetic Pathways; Carotenoids; Dioxygenases; Germination; Isomerases; Lactones; Molecular Structure; Mutation; Oryza; Phenotype; Pisum sativum; Plant Growth Regulators; Plant Proteins; Stereoisomerism; Striga

The gene encoding human β-carotene-9',10'-oxygenase, which cleaves the 9',10' double bond in β-carotene into β-apo-10'-carotenal, was cloned and expressed in Escherichia coli. Under aqueous conditions, the optimum organic solvent for the formation of detergent micelles was toluene. The optimum pH, temperature, detergent type, and the optimum concentrations of detergent, substrate, and enzyme for β-apo-10'-carotenal production were 8.0, 37°C, Tween 40, 2.4%, 300 mg β-carotene/l, and 0.25 U/ml, respectively. Under the optimum conditions, 43 mg β-apo-10'-carotenal/l was produced after 21 h with a conversion of 14%. This is the first report to describe the enzymatic production of β-apo-10'carotenal.

Topics: beta Carotene; Biotechnology; Carotenoids; Cloning, Molecular; Detergents; Dioxygenases; Escherichia coli; Fatty Acid Desaturases; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Micelles; Recombinant Proteins; Solvents; Temperature

Extracellular liquid of the edible fungus Lepista irina was found to effectively degrade beta,beta-carotene, beta-lonone, beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed as volatile breakdown products of beta,beta-carotene with mycelium-free culture supernatants, whereas beta-apo-10'-carotenal was identified as non-volatile degradation product. The key enzyme catalyzing the oxidative cleavage of beta,beta-carotene was purified with an overall yield of 63% and a purification factor of 43. Biochemical characterization showed a molecular mass of 50.5 kDa and an isoelectric point of 3.75. Fastest beta,beta-carotene degradation occurred at 34 degrees C and pH values between 3.5 and 4. Degenerate oligonucleotides were derived from N-terminal and internal amino acid sequences. By means of PCR-based cDNA-library screening a 1284 bp cDNA was identified which showed great overall similarity to Pleurotus eryngii polyvalent peroxidases. The obtained sequence contains an open reading frame of 1083 nucleotides, encoding a polypeptide of 361 amino acids. A 30 amino acid signal peptide was identified upstream of the N-terminal sequence of the mature enzyme. The L. irina versatile peroxidase represents the first microbial enzyme capable of carotenoid degradation that has been characterized on a molecular level, proving the participation of extracellular enzymes of white rot fungi in biotic carotenoid degradation processes.

Topics: Amino Acid Sequence; Basidiomycota; beta Carotene; Biodegradation, Environmental; Carotenoids; Culture Media; Flavoring Agents; Hydrogen-Ion Concentration; Molecular Sequence Data; Norisoprenoids; Oxidation-Reduction; Peroxidase; Temperature; Time Factors; Volatilization

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Topics: Amino Acid Sequence; Animals; beta Carotene; beta-Carotene 15,15'-Monooxygenase; Carotenoids; Catalysis; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA, Complementary; Drosophila; Drosophila Proteins; Expressed Sequence Tags; Female; Gene Library; Humans; Lycopene; Male; Mass Spectrometry; Mice; Mice, Inbred BALB C; Models, Chemical; Molecular Sequence Data; Norisoprenoids; Oxygen; Oxygenases; Phenotype; Phylogeny; Retinaldehyde; RNA; Sequence Homology, Amino Acid; Terpenes; Time Factors; Tissue Distribution; Vitamin A; Zebrafish