Correlation of the content of hepatotoxin nodularin and glycosidic carotenoids, 4-ketomyxol-2′-fucoside and novel 1′-O-methyl-4-ketomyxol-2′-fucoside,in 20 strains of the cyanobacterium Nodularia spumigena

The carotenoids of 19 different strains of Nodularia spumigena and one Nodularia sphaerocarpa from different global locations were investigated. The molecular structure of the diagnostic pigment in N. spumigena of the Baltic Sea, tentatively named ‘4-keto-myxoxanthophyll-like pigment’ in Schlüter, L., Garde, K., Kaas, H., [2004. A 4-keto-myxoxanthophyll-like pigment is a diagnostic pigment for the toxic cyanobacteria Nodularia spumigena in the Baltic Sea. Mar. Ecol. Prog. Ser. 275, 69–78.] was determined to be a 4-ketomyxol-2′-fucoside. In most of the strains an additional carotenoid was found, identified as the novel 1′-O-methyl-4-ketomyxol-2′-fucoside by 2D NMR. This glycosidic carotenoid methyl ether was found to be a more important diagnostic pigment than the 4-ketomyxol-2′-fucoside for the toxic N. spumigena in the Baltic Sea. Out of the 20 strains 15 were found to produce the hepatotoxin nodularin. The content of carotenoids and nodularin was found to increase relative to chlorophyll a at increasing light intensity and at stationary growth, and nodularin was significantly correlated to both 4-ketomyxol-2′-fucoside and 1′-O-methyl-4-ketomyxol-2′-fucoside, and particular to the sum of these two pigments.     

A crtA-related gene from Flavobacterium P99-3 encodes a novel carotenoid 2-hydroxylase involved in myxol biosynthesis

A genomic DNA fragment with carotenogenic genes involved in myxol biosynthesis (3′,4′-didehydro-1′,2′-dihydro-β,ψ-carotene-3,1′,2′-triol) was cloned from Flavobacterium P99-3. It contains a gene highly homologous to crtA from purple bacteria encoding there an acyclic carotenoid 2-ketolase. Since no ketolation step is involved in myxol biosynthesis, the function of crtA-OH from Flavobacterium was assigned by complementation in Escherichia coli engineered to synthesize demethylspheroidene and 1′-hydroxy-demethylspheroidene. Upon co-expression of crtA-OH, the formation of 2-hydroxy derivatives of both carotenoids assigns CrtA-OH as a novel carotenoid hydroxylase. The gene was used to re-construct myxol biosynthesis in E. coli successfully. Additionally, 1′,2′-dihydroxytorulene and 1,2,1′-trihydroxy-3,4,3′,4′-tetradehydrolycopene were obtained. Their generation demonstrates that a new class of 2-hydroxy carotenoids can now be pursued by genetic engineering in E. coli.     

The gene carD encodes the aldehyde dehydrogenase responsible for neurosporaxanthin biosynthesis in Fusarium fujikuroi

Neurosporaxanthin (β-apo-4'-carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β-apo-4'-carotenal. On the basis of sequence homology to Neurospora crassa YLO-1, which mediates the formation of apo-4'-lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β-apo-4'-carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid-overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β-apo-4'-carotenal, its derivative β-apo-4'-carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the ΔcarD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo-1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid-overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established.     

The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway

The accumulation of the apocarotenoid neurosporaxanthin and its carotene precursors explains the orange pigmentation of the Neurospora surface cultures. Neurosporaxanthin biosynthesis requires the activity of the albino gene products (AL-1, AL-2 and AL-3), which yield the precursor torulene. Recently, we identified the carotenoid oxygenase CAO-2, which cleaves torulene to produce the aldehyde β-apo-4'-carotenal. This revealed a last missing step in Neurospora carotenogenesis, namely the oxidation of the CAO-2 product to the corresponding acid neurosporaxanthin. The mutant ylo-1 , which exhibits a yellow colour, lacks neurosporaxanthin and accumulates several carotenes, but its biochemical basis is unknown. Based on available genetic data, we identified ylo-1 in the Neurospora genome, which encodes an enzyme representing a novel subfamily of aldehyde dehydrogenases, and demonstrated that it is responsible for the yellow phenotype, by sequencing and complementation of mutant alleles. In contrast to the precedent structural genes in the carotenoid pathway, light does not induce the synthesis of ylo-1 mRNA. In vitro incubation of purified YLO-1 protein with β-apo-4'-carotenal produced neurosporaxanthin through the oxidation of the terminal aldehyde into a carboxyl group. We conclude that YLO-1 completes the set of enzymes needed for the synthesis of this major Neurospora pigment.     

Production of caloxanthin 3'-β-D-glucoside, zeaxanthin 3,3'-β-D-diglucoside, and nostoxanthin in a recombinant Escherichia coli expressing system harboring seven carotenoid biosynthesis genes, including crtX and crtG

The aim of this study was to produce rare β-carotene-modified carotenoids possessing 2-O (-H or -glu) and/or 3-O (-H or -glu) functionalities in their β-ionone ring(s) using a recombinant Escherichia coli approach. This involved expressing seven carotenoid biosynthesis genes (crtE, crtB, crtI, crtY, crtZ, crtX and crtG). From the cells of the recombinant E. coli, caloxanthin (β,β-carotene-2,3,2′,3′-tetrol)-3′-β-d-glucose, zeaxanthin (β,β-carotene-3,3′-diol) 3,3′-β-d-diglucoside, and nostoxanthin (β,β-carotene-2,3,3′-triol) (rare carotenoids) were isolated and identified. Caloxanthin 3′-β-d-glucose displayed potent ¹O₂ quenching activity (IC₅₀ 19 μM).     

Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and β-cryptoxanthin by ferret carotene-9',10'-monooxygenase

Xanthophyll carotenoids, such as lutein, zeaxanthin and β-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,15′-monooxygenase (CMO1) has been shown to be involved in vitamin A formation, while recent studies suggest that carotene-9′,10′-monooxygenase (CMO2) may have a broader substrate specificity than previously recognized. In this in vitro study, we investigated baculovirus-generated recombinant ferret CMO2 cleavage activity towards the carotenoid substrates zeaxanthin, lutein and β-cryptoxanthin. Utilizing HPLC, LC–MS and GC–MS, we identified both volatile and non-volatile apo-carotenoid products including 3-OH-β-ionone, 3-OH-α-ionone, β-ionone, 3-OH-α-apo-10′-carotenal, 3-OH-β-apo-10′-carotenal, and β-apo-10′-carotenal, indicating cleavage at both the 9,10 and 9′,10′ carbon–carbon double bond. Enzyme kinetic analysis indicated the xanthophylls zeaxanthin and lutein are preferentially cleaved over β-cryptoxanthin, indicating a key role of CMO2 in non-provitamin A carotenoid metabolism. Furthermore, incubation of 3-OH-β-apo-10′-carotenal with CMO2 lysate resulted in the formation of 3-OH-β-ionone. In the presence of NAD⁺, in vitro incubation of 3-OH-β-apo-10′-carotenal with ferret hepatic homogenates formed 3-OH-β-apo-10′-carotenoic acid. Since apo-carotenoids serve as important signaling molecules in a variety of biological processes, enzymatic cleavage of xanthophylls by mammalian CMO2 represents a new avenue of research regarding vertebrate carotenoid metabolism and biological function.     

Characterization of an apo-carotenoid 13,14-dioxygenase from Novosphingobium aromaticivorans that converts β-apo-8′-carotenal to β-apo-13-carotenone

A putative carotenoid oxygenase from Novosphingobium aromaticivorans was purified with a specific activity of 0.8?U/mg by His-Trap affinity chromatography. The native enzyme was estimated to be a 52?kDa monomer. Enzyme activity for β-apo-8′-carotenal was maximal at pH 8.0 and 45?°C, with a half life of 15.3?h, K _m of 21?μM, and k _cat of 25?l/min. The enzyme exhibited cleavage activity only for carotenoids containing one β-ionone ring and its catalytic efficiency (k _cat/K _m) followed the order β-apo-8′-carotenal?>?β-apo-4′-carotenal?>?γ-carotene. The enzyme converted these carotenoids to β-apo-13-carotenones by cleaving their C₁₃–C₁₄ double bonds. The oxygen atom of β-apo-13-carotenone originated not from water but from molecular oxygen. Thus, the enzyme was an apo-carotenoid 13,14-dioxygenase.     

In vitro and in vivo characterization of retinoid synthesis from beta-carotene

Retinoids are indispensable for the health of mammals, which cannot synthesize retinoids de novo. Retinoids are derived from dietary provitamin A carotenoids, like β-carotene, through the actions of β-carotene-15,15′-monooxygenase (BCMO1). As the substrates for retinoid-metabolizing enzymes are water insoluble, they must be transported intracellularly bound to cellular retinol-binding proteins. Our studies suggest that cellular retinol-binding protein, type I (RBP1) acts as an intracellular sensor of retinoid status that, when present as apo-RBP1, stimulates BCMO1 activity and the conversion of carotenoids to retinoids. Cellular retinol-binding protein, type II (RBP2), which is 56% identical to RBP1 does not influence BCMO1 activity. Studies of mice lacking BCMO1 demonstrate that BCMO1 is responsible for metabolically limiting the amount of intact β-carotene that can be absorbed by mice from their diet. Our studies provide new insights into the regulation of BCMO1 activity and the physiological role of BCMO1 in living organisms.     

Sterically hindered carotenoids with 3Z, 5Z configuration from the seeds of oriental bitter sweet, Celastrus orbiculatus

Sterically hindered cis-carotenoids 1 and 2 were isolated from seeds of the oriental bitter sweet, Celastrus orbiculatus. Their structures were determined to be (3′Z, 5′Z)-celaxanthin and (3′Z, 5′Z)-torulene, respectively, on the basis of spectroscopic data and iodine-catalyzed stereomutation. This is the first report on carotenoids with a 3Z, 5Z configuration.     

Relationship between cyclic AMP level and accumulation of carotenoid pigments in Neurospora crassa

Dark grown mycelial cells of Neurospora crassa bearing mutant genes crisp-I or frost and having a decreased level of cyclic adenosine 3,5-monophosphate contained more carotenoid pigments than the cells with wild alleles of these genes. A transient decrease of the cyclic AMP occurred following photoinduction of carotenoid synthesis during its lag-period. Its intensity correlated with the increase of carotenoid pigment level due to photoinduction. No correlation in the content of cyclic guanosine 5-phosphate with both constitutive level of carotenoids and its photoinduced increase was observed.     

Carotenoids of Anacystis nidulans,structures of caloxanthin and nostoxanthin

Reinvestigation of the carotenoids of Anacystis nidulans has confirmed the occurrence of β,β-carotene (β-carotene), β,β-caroten-3-ol (cryptoxanthin), β,β-carotene-3,3′-diol (zeaxanthin) and 2R,3R,3′R-β,β-carotene-2,3,3′-triol (absolute configuration assigned in the present work). In addition the previously unknown 2R,3R,2′R,3′R-β,β-carotene-2,3,2′,3′-tetrol has been isolated. The triol and the tetrol are considered identical with caloxanthin and nostoxanthin, respectively, for which allenic structures have been suggested by others. The chirality of these compounds followed from CD and ¹H NMR considerations.     

The carotenoid pigments in the juice and flavedo of a mandarin hybrid (Citrus reticulata) cv michal during ripening

The carotenoid pigments ofthe mandarin hybrid (Citrus reticulata) cv Michal, in the juice and flavedo were characterized at three ripening stages, before, during and after colour break. During ripening the characteristic mandarin pattern was formed in the juice, which contained cryptoxanthin as the principal pigment. In the flavedo the chloroplast carotenoid pattern of the green fruit changed into the characteristic pattern of C. reticulata with a high level of citraurin which, together with cryptoxanthin, imparts an intensive reddish tint to the hybrid. The flavedo contained an unusual C₃₀ apocarotenoid, β-citraurinene (8′-apo-β-caroten-3-ol). The flavedo carotenoids of this accidental hybrid were compared with the carotenoids of the presumed parents Dancy tangerine and Clementine. The hybrid resembles more the second parent, from which it inherited the ability to biosynthesize a higher amount of citraurin as well as citraurinene. Citraurinene, considered a Citrus hybrid-specific pigment, was found for the first time in a Citrus variety. A possible biosynthetic pathway of the Citrus C₃₀ -apocarotenoids is proposed.     

In situ hydrothermal syntheses, crystal structures and luminescent properties of two novel zinc(II) coordination polymers based on tetrapyridyl ligand

Two novel Zn(II) coordination polymers, [Zn(2-pytpy)(fum)]_n·nH₂O (1) and [Zn₆(4-pytpy)₃(mal)₄]_n·5n(H₂O) (2), (2-pytpy = 4′-(4-pyridyl)-2,2′:6′,2″-terpyridine, 4-pytpy = 4′-(4-pyridyl)-4,2′:6′,4″-terpyridine, H₂fum = fumaric acid and H₂mal = malic acid) have been hydrothermally synthesized and structurally characterized. Notably, in situ ligand reactions occur in the formation of complexes 1 and 2, in which maleic acid is converted into fumaric acid and malic acid, respectively. Complex 1 is a 1D infinite chain structure, which is extended into a supramolecular layer by intermolecular π…π stacking interactions. Complex 2 is a 3D network structure, in which the bidentate-bridging 4-pytpy ligands link the layers based on the tetranuclear Zn(II) subunits to form the (4,10)-connected network. The luminescent properties of 1 and 2 have been investigated with emission spectra and UV-Vis diffuse reflectance spectra in the solid state. Additionally, these two complexes possess great thermal stabilities.     

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis,chemiluminescence, and DNA damage analyses

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1ΔcrtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H₂O₂ and singlet oxygen than two carotenes (lycopene and β-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1′ position, compared with other tested carotenoids.     

Activity of E. coli ClpA bound by nucleoside diphosphates and triphosphates

The Escherichia coli ClpA protein is a molecular chaperone that binds and translocates protein substrates into the proteolytic cavity of the tetradecameric serine protease ClpP. In the absence of ClpP, ClpA can remodel protein complexes. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleoside triphosphate binding to first assemble into a hexamer. Here we report the assembly properties of ClpA in the presence of the nucleoside diphosphates and triphosphates ADP, adenosine 5′-[γ-thio]triphosphate, adenosine 5′-(β,γ-imido)triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and adenosine diphosphate beryllium fluoride. In addition to examining the assembly of ClpA in the presence of various nucleotides and nucleotide analogues, we have also correlated the assembly state of ClpA in the presence of these nucleotides with both polypeptide binding activity and enzymatic activity, specifically ClpA-catalyzed polypeptide translocation. Here we show that all of the selected nucleotides, including ADP, promote the assembly of ClpA. However, only adenosine 5′-[γ-thio]triphosphate and adenosine 5′-(β,γ-imido)triphosphate promote the formation of an oligomer of ClpA that is active in polypeptide binding and translocation. These results suggest that the presence of γ phosphate may serve to switch ClpA into a conformational state with high peptide binding activity, whereas affinity is severely attenuated when ADP is bound.     

Sodium borohydride reduction of anthocyanidins

The reduction of anthocyanidins with NaBH₄ in EtOH or MeOH produces inter alia racemates of epicatechins. Thus, the (±) racemates of 3′,5′-di-O-methylepigallocatechin, 3′-O-methylepigallocatechin, and 3′-O-methylepicatechin have been identified as the reduction products of malvidin, petunidin, and peonidin, respectively, by their UV, MS and NMR spectra.     

Coincident isolation of a novel homoisoflavonoid from Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae)

We report on the first phytochemical investigation of a member of the African genus Resnova (Hyacinthoideae: Hyacinthaceae). From the dichloromethane extract of the bulbs of both Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae) a novel 3-benzyl-4-chromanone homoisoflavonoid, 5,6-dimethoxy-7-hydroxy-3-(4′-hydroxybenzyl)-4-chromanone, was isolated. A further 11 known homoisoflavonoids were also identified, the 12 in total presenting a clear biosynthetic sequence. Eight of the 12 compounds found were common to both species.     

Are carotenoids the blue-light photoreceptor in the photoinduction of protoperithecia in Neurospora crassa?

A triple albino mutant of Neurospora crassa with a measured content of carotenoids absorbing at 470 nm less than 0.5% of that of the wild type (calculated value less than 8·10^-4%) had the same threshold for photoinduction of protoperithecia as the wild type when illuminated with monochromatic light at 471 nm. This is strong evidence against the hypothesis that the bulk of carotenoids are the blue-light photoreceptor for this phenomenon. However, it is impossible to exclude traces of carotenoids acting as the photoreceptor at less than 3·10^-12 M in a very efficient sensory transduction chain.Abbreviations A absorbance - al albino mutant - WT wild type