Β-end Groups Research Articles

Microalgal AstaP is a natural solubilizer of astaxanthin with broad binding ability for carotenoids and related compounds

Astaxanthin-binding water-soluble proteins, named AstaPs, were identified as photooxidative-stress inducible aqueous carotenoprotein found in microalgae of the family Scenedesmaceae. Although AstaPs bind astaxanthin, the properties of selective binding of astaxanthin remain unclear. In this study, we prepared carotenoid-free apo-AstaPs and investigated the ability to bind carotenoids. Apo-AstaP-orange1 selectively bound carotenoids, especially astaxanthin, from the pigments extracted from stressed cells containing secondary carotenoids, chlorophylls, and β-carotene. Reconstitution assays revealed that apo-AstaP-orange1 showed higher affinity for binding astaxanthin than the other tested carotenoids. Other AstaP family proteins, such as AstaP-orange2 and AstaP-pink1 from Scenedesmus obtusus Oki-4N, and recombinant AstaP-pink1 expressed in E. coli, also displayed a preference for binding astaxanthin. Apo-AstaP-orange1 bound astaxanthin and astaxanthin-monoesters but not astaxanthin-diesters, indicating that the protein requires at least one non-esterified β-end group for binding of the carotenoids. On mixing in vitro, apo-AstaP-orange1 also had the ability to bind lutein, adonixanthin, canthaxanthin, and other tested xanthophylls and carotenoid derivatives such as retinoids. Our results indicate that AstaPs are selective solubilizers of astaxanthin in vivo and potential natural solubilizers of carotenoids and carotenoid derivatives in vitro.

Metabolism of Carotenoids in Mammals.

Pathways for xanthophyll metabolism have been proposed on the basis of several oxidation products of dietary xanthophylls detected in the tissues of fish, birds, and human subjects. No enzyme reaction had been characterized as responsible for the pathways until a mouse liver homogenate was found to oxidize the 3-hydroxy β-end of xanthophylls to a 3-oxo ε-end in the presence of a cofactor, NAD+. This oxidation consists of dehydrogenation to an unstable intermediate having a 3-oxo β-end group and the subsequent migration of a double bond. β,ε-Caroten-3'-one, a metabolite of β-cryptoxanthin, was found in human plasma, indicating that the same oxidative activity as that found in the mouse liver works in human tissues.The oxidative cleavage of carotenoids is mediated by two dioxygenases: a central cleavage enzyme and an asymmetric cleavage enzyme. In mice, the latter enzyme was suggested to eliminate carotenoids in tissues, while in humans, this enzyme is inactivated, resulting in carotenoid accumulation. In this chapter, carotenoid metabolism in mammals is described in terms of the oxidation of functional groups and cleavage of the carbon skeleton.

Inactivation of bciD and bchU genes in the green sulfur bacterium Chlorobaculum limnaeum and alteration of photosynthetic pigments in the resultant mutants

Abstract The bciD and bchU genes were earlier identified as relating to the C7-formylation and to catalyzing the C20-methylation, respectively, of bacteriochlorophyll(BChl)- e biosynthesis in the green sulfur bacterium Chlorobaculum ( Cba .) limnaeum . The resultant Δ bciD and Δ bchU mutants produced BChl- c and BChl- f , respectively, as the BChl pigments in the cultured cells. In this study, the mutant deleting both the genes was constructed and successfully accumulated BChl- d in the same species. The series of the mutants using the Cba . limnaeum RK-j-1 strain enabled us to synthesize the four different BChl pigments, BChls- c , d , e , and f , in the identical species. In order to evaluate photo-adaptation mechanisms of green sulfur bacteria, alteration of photosynthetic pigments including carotenoids (Cars) by the mutation as well as illuminated light intensity was investigated using HPLC. For the BChl pigments, inactivation of the bciD gene induced drastic changes in the composition of BChl homologs (further methylation at the 8 2 -position from the 8-ethyl to isobutyl group) and epimers (inversion of the 3 1 -stereochemistry from (3 1 R )- to (3 1 S )-configuration) compared to inactivation of the bchU gene; the total contents of the (3 1 S )-epimers accounted for 23–36% in the wild type strain and for 87–90% in the Δ bciD / bchU double mutant, depending on illuminated light intensity. Such mutation did not affect Car biosynthesis, but the composition was strongly light-dependent. With an increase of illuminated light intensity, Cars possessing aromatic ϕ-end group(s), isorenieratene and β-isorenieratene that were characteristic of the Cba. limnaeum species, decreased for all three mutants as well as the wild type strain. Concomitantly, the contents of Cars possessing β-end groups, β-carotene and 7,8-dihydro-β-carotene, increased and were dominant under high-light illumination.

A 3-hydroxy %-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals

We previously found that mice fed lutein accumulated its oxidative metabolites (3'-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3'-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD(+), (3'R,6'R)-3'-hydroxy-β,ε-caroten-3-one and (6RS,3'R,6'R)-3'-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3' and C6' of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3'-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.

Structure–response relationship of carotenoid bioaccessibility and antioxidant activity as affected by the hydroxylation and cyclization of their terminal end groups

The structurally related carotenoids β-carotene, γ-carotene, lycopene, rubixanthin, and β-cryptoxanthin differ in their terminal end groups and the presence or absence of a hydroxyl function. In this study, emulsions containing these carotenoids were subjected to an in vitro digestion to compare bioaccessibilities and antioxidant activities of the resulting micellar fractions. The bioaccessibility of lycopene and β-carotene was 28.6±0.1% and 35.7±0.3% respectively, γ-carotene bioaccessibility was between these values (32.7±0.2%), while the bioaccessibility of rubixanthin (21.8%) and β-cryptoxanthin (28.8%) was significantly lower. The antioxidant activity of the corresponding micellized carotenoid fractions ranked as follows: rubixanthin≥lycopene>β-cryptoxanthin>γ-carotene>β-carotene for both the TEAC and FRAP assays. A similar trend was observed for the hexane-dissolved carotenoids. A negative linear correlation between bioaccessibility and FRAP (R2=0.7395) and TEAC (R2=0.9125) values was established. The presence of both open ψ-end groups and hydroxyl functions led to higher antioxidant activities as compared to the unsubstituted β-end group, presumably resulting in decreased stability during digestion and, thus, lowered bioaccessibility.

Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids).

Chromatographic performance on a C 30-bonded stationary phase of monohydroxycarotenoids with variable chain length or degree of desaturation and of lycopene isomers synthesized by various carotene desaturases

Selectivity towards geometric isomers is a superior feature of a C 30 polymeric stationary phase. Therefore, lycopene isomers synthesized in Escherichia coli transformants by catalysis of divers carotene desaturases were separated on this stationary phase. Due to their spectral characteristics and by co-chromatography with nuclear magnetic resonance-characterized carotene standards, some of them could be identified. Most of the lycopene isomers were cyclized by lycopene cyclase yielding mainly 9 Z, 13 Z and all- E β-carotene. In contrast, 7,9,7′,9′ Z prolycopene is accumulating since it cannot be converted by this enzyme. Finally several acyclic hydroxycarotenoids with a chain of 30, 40 and 45 carbon atoms differing in the length of the polyene chain from 9 to 13 were separated on the C 30 stationary phase. Longer retention times were observed when the length of the molecule increased and also when the conjugated double bond system was extended. Corresponding monocyclic carotenoids were less retained on the C 30 stationary phase and derivatives with an ϵ-ionone end group eluted earlier than with a β-end group.

Molecular and Crystal Structures of 2-(All-trans-Retinylidene)-Indan-1,3-Dione

Molecular structures of a novel carotenoid analogue, 2-(all-trans-retinylidene)-indan-1,3-dione (RetInd), were determined in solution and in the crystalline state by 1H-NMR spectroscopy and by X-ray crystallography, respectively. They were compared with those of all-trans-retinal (Ret), a visual chromophore. Ret takes a 6s-cis configuration (a cis configuration around the C6–C7 single bond) that is usually found in carotenoid analogues that have β-end groups, both in solution and in the crystalline state. On the other hand, it was found that RetInd took a 6s-cis configuration in solution but it took a 6s-trans configuration in the crystalline state. Comparison of the crystallographic structures between Ret and RetInd lead us to the conclusion that a van der Waals attraction in polyene chains and steric repulsion between a cyclohexene and an indan-1,3-dione ring stabilized the 6s-trans configuration of RetInd in the crystalline state.

Botryoxanthin B and α-botryoxanthin A from the green microalga Botryococcus braunii Kawaguchi-1

Two new carotenoids, botryoxanthin B and α-botryoxanthin A, were isolated from the Kawaguchi-1 strain of the green microalga Botryococcus braunii in addition to botryoxanthin A. They had 4-keto-β and ε-end groups instead of the β-end group present in botryoxanthin A.

Metabolism of Carotenoids in the Apple Snail, Pomacea canaliculata

Feeding experiments with β,β-carotene, canthaxanthin, (3R,3′R)-zeaxanthin, (3R,3′R,6′R)-lutein and racemic astaxanthin on the apple snail Pomacea canaliculata were investigated. Based on the experimental results, β,β-carotene was oxidatively metabolized, and the resulting principal metabolic product (3S,3′S)-astaxanthin along with other keto carotenoids such as (3S)-3-hydroxy-β-echinenone, canthaxanthin, (3S,3′R)-4-ketozeaxanthin and (3S)-phoenicoxanthin was accumulated in the gonad. Similarly, (3R,3′R,6′R)-lutein was converted into fritschiellaxanthin [(3S,3′R,6′R)-3,3′-dihydroxy-β, ϵ-caroten-4-one]. It was concluded that P. canaliculata had the ability to oxidize the C-3, 4 positions on the β-end group.

Silver ion spray reagent for the discrimination of β- and ε-end groups in carotenoids on thin-layer chromatograms

Thin-layer chromatograms of pure carotenoids and natural carotenoid mixtures were dried and sprayed with saturated methanolic silver nitrate solution. Those carotenoids containing β-end groups showed a marked bathchromic colour change for each such end group. Thus, β-carotene, β-cryptoxanthin and zeaxanthin gave a clear red colour while α-carotene, α-cryptoxanthin and lutein gave deep organge spots after such treatment. Taraxanthin and antheraxanthin could be similarly distinguished. The method was not applicable to paper chromatography.

Studies on the carotenoids in the muscle of salmon-II. Astaxanthin and/or canthaxanthin-actomyosin complex in salmon muscle.

It was proposed that astaxanthin and/or canthaxanthin in salmon muscle binds with one β-ionone ring to a hydrophobic binding site which exists on the surface of actomyosin. The binding of carotenoids to actomyosin is not specific. The hydroxyl and keto groups of β-end group intensify the binding strength of carotenoids to actomyosin.

Carotenoids in food chain studies—III. Resorption and metabolic transformation of carotenoids in Mytilus edulis (Edible mussel)

Abstract 1. 1. The quantitative carotenoid composition of M. edulis from two geographical localities at different seasons is reported. 2. 2. Twenty different carotenoids are structurally identified. Carotenoids with an acetylenic 3-hydroxy-7,8-didenhydro β-end group accumulate in M. edulis . 3. 3. Feeding experiments with algal unicultures are reported. 4. 4. Fucoxanthin, 19′-hexanoyloxyfucoxanthin, diadinoxanthin and peridinin were resorbed. 5. 5. Hydrolysis of carotenoid acetates, conversion of allenic to acetylenic end groups and conversion of 5,6-epoxides to 5,6-glycols were general metabolic reactions. 6. 6. Fucoxanthin provides isomytiloxanthin, confirmed by conversion of 19′-hexanoyloxyfucoxanthin to 19′-hexanoyloxyisomytiloxanthin. 7. 7. Fucoxanthin is converted to halocynthiaxanthin. 8. 8. The diacetylenic alloxanthin is a terminal metabolic product. 9. 9. Peridinin is converted to peridininol and pyrrhoxanthinol. 10. 10. Metabolic schemes are proposed.

Synthesen von optisch aktiven Carotinoiden mit einer (R)‐4‐Hydroxy‐β‐Endgruppe

Synthesis of Optically Active Carotenoids with (R)‐4‐Hydroxy β‐End GroupsWe describe the synthesis of optically active iso‐β‐kryptoxanthin (12; (R)‐β,β‐caroten‐4‐ol), iso‐α‐kryptoxanthins 14 ((4R,6′RS)‐β,ε‐caroten‐4‐ol) and 16 ((4R,6′R)‐β,ε‐caroten‐4‐ol), 4′‐hydroxyechinenone (18; (R)‐4′‐hydroxy‐β,β‐caroten‐4‐one), and isorubixanthin (20; (R)‐β,ω,‐caroten‐4‐ol), their 400‐MHz‐1H‐NMR spectra, CD spectra and HPLC behaviour.

Direct diastereomeric resolution of carotenoids : I. 3-hydroxy-4-ocy β-end group

Direct diastereomeric resolution of carotenoids : I. 3-hydroxy-4-ocy β-end group