Biosynthesis of 9-cis-Retinoic Acid from 9-cis-β-Carotene in Human Intestinal Mucosa in Vitro

The role of 9-cis-β-carotene (9-cis-β-C) as a potential precursor of 9-cis-retinoic acid (9-cis-RA) has been examined in human intestinal microcosa in vitro. By using HPLC, uv spectra, and chemical derivatization analysis, both 9-cis-RA and all-trans-retinoic acid (all-trans-RA) have been identified in the postnuclear fraction of human intestinal microcosa after incubation with 9-cis-β-C at 37°C. The biosynthesis of both 9-cis-RA and all-trans-RA from 9-cis-β-C was linear with increasing concentrations of 9-cis-β-C (2-30 μM) and was linear with respect to tissue protein concentration up to 0.75 mg/ml. Retinoic acid was not detected when a boiled incubation mixture was incubated in the presence of 9-cis-β-C. The rate of synthesis of 9-cis- and all-trans-RA from 4 μM 9-cis-β-C were 16 ± 1 and 18 ± 2 pmol/hr/mg of protein, respectively. However, when 2 μM all-trans-β-C was added to the 4 μM 9-cis-β-C, the rate of all-trans-RA synthesis was increased to 38 ± 6 pmol/hr/mg of protein, whereas the rate of 9-cis-RA synthesis remained the same. These results suggest that 9-cis-RA is produced directly from 9-cis-β-C. Furthermore, incubations of either 0.1 μM 9-cis- or all-trans-retinal under the same incubation conditions showed that 9-cis-RA could also arise through oxidative conversion of 9-cis-retinal. Although only 9-cis-RA was detected when 9-cis-RA was used as the substrate, the isomerization of the all-trans-RA to 9-cis-RA cannot be ruled out, since both all-trans-RA and trace amounts of 9-cis-RA were detected when all-trans-retinal was incubated as the substrate. These data indicate that 9-cis-β-C can be a source of 9-cis-RA in the human. This conversion may have a significance in the anticarcinogenic action of β-C.     

Substrate Specificity and Subcellular Localization of the Aldehyde-Alcohol Redox-coupling Reaction in Carp Cones

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.     

Configuration of the carotenoid in the reaction centers of photosynthetic bacteria. Comparison of the resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C with those of cis-trans isomers of β-carotene

The resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C as well as those of the cis-trans isomers of β-carotene (all-trans, 9-cis, 13-cis, 15-cis and 9-cis, 13-cis- (or 9-cis, 13′-cis)) have been recorded at liquid N₂ temperature by use of the 457.9, 488.0 and 514.5 nm excitation lines. Comparison of the spectra indicated that the carotenoid in the reaction center takes the 15-cis configuration.     

cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.     

Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.     

Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

Background

Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.

Methods

We have determined apparent dissociation constants, K′_d, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.

Results

CRBPI and CRBPII, respectively, bind 9-cis-retinol (K′_d, 11 nM and 68 nM) and 9-cis-retinal (K′_d, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.

Conclusions

CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.

General significance

These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.     

The Action of 11-cis-Retinol on Cone Opsins and Intact Cone Photoreceptors

11-cis-Retinol has previously been shown in physiological experiments to promote dark adaptation and recovery of photoresponsiveness of bleached salamander red cones but not of bleached salamander red rods. The purpose of this study was to evaluate the direct interaction of 11-cis-retinol with expressed human and salamander cone opsins, and to determine by microspectrophotometry pigment formation in isolated salamander photoreceptors. We show here in a cell-free system using incorporation of radioactive guanosine 5′-3-O-(thio)triphosphate into transducin as an index of activity, that 11-cis-retinol inactivates expressed salamander cone opsins, acting an inverse agonist. Similar results were obtained with expressed human red and green opsins. 11-cis-Retinol had no significant effect on the activity of human blue cone opsin. In contrast, 11-cis-retinol activates the expressed salamander and human red rod opsins, acting as an agonist. Using microspectrophotometry of salamander cone photoreceptors before and after bleaching and following subsequent treatment with 11-cis-retinol, we show that 11-cis-retinol promotes pigment formation. Pigment was not formed in salamander red rods or green rods (containing the same opsin as blue cones) treated under the same conditions. These results demonstrate that 11-cis-retinol is not a useful substrate for rod photoreceptors although it is for cone photoreceptors. These data support the premise that rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type. These mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.11-cis-Retinol is the precursor to 11-cis-retinal, the 11-cis-aldehyde form of vitamin A and the chromophore that combines covalently with rod and cone opsin proteins to form visual pigments. 11-cis-Retinal is consumed during visual signaling, and its continual synthesis is required. Photon absorption by the visual pigments causes the isomerization of its chromophore to the all-trans configuration. This initiates two processes critical for vision: activation of the photoreceptor cell and the eventual recovery of the original photosensitivity of the cells, requiring regeneration of the visual pigments. As cones are used for bright light vision, these two processes must work more rapidly in cones than in rods and thus cones have a higher requirement of 11-cis-retinoids as suggested by Rushton (1, 2).Photoreceptor activation begins with photoisomerization of the chromophore within the visual pigment. This results in a subsequent conformational change of the protein part of the visual pigment that is able to activate its G protein transducin, which in turn activates a PDE that lowers the concentration of cGMP and closes cGMP-gated ion channels. These steps comprise the visual signal transduction cascade (see Ref. 3 for review).The visual cycle involves regeneration of the visual pigment, which ultimately deactivates the protein and accomplishes the recovery of the photosensitivity of the photoreceptor cell. Classically, this process involves both the photoreceptor cell and the retinal pigment epithelium (RPE).⁴ After photoisomerization of the chromophore and formation of the active visual pigment, all-trans-retinal is released from the opsin and reduced to all-trans-retinol, which is then transported to the RPE where it is isomerized to 11-cis-retinol through a number of steps. In the RPE, 11-cis-retinol is oxidized to the aldehyde form, which is transported back to the photoreceptor cell and can be directly used by all of the opsins to regenerate an inactive pigment ready for photoactivation. The details of this model have been extensively reviewed (4, 5). Alternatively, recent work suggests that cones have an additional source of 11-cis-retinoids from Müller cells (6–8). Like the RPE cells, Müller cells have been shown to be able to convert all-trans-retinol to 11-cis-retinol (6). Unlike in the RPE cells, 11-cis-retinol is not oxidized to 11-cis-retinal in Müller cells.Jones et al. (9) demonstrated that administration of 11-cis-retinol to bleached salamander red cones could restore photosensitivity. A logical conclusion was that red cones were able to oxidize 11-cis-retinol to the aldehyde and regenerate visual pigments although noncovalent binding of 11-cis-retinol to red cone opsins generating a light-sensitive complex could not be excluded. On the other hand, 11-cis-retinol does not restore photosensitivity to bleached salamander rod cells but appears to directly activate the cells (9, 10). The data suggested that the rods were not able to oxidize 11-cis-retinol, but that the retinol itself could activate the signal transduction cascade, and indeed we recently demonstrated that 11-cis-retinol acts as an agonist to expressed bovine rod opsin (11). Our aim here was to study the action of 11-cis-retinol on cone opsins and cone photoreceptor cells to determine the efficacy of an alternate visual cycle for cones.The photoreceptor cells used in this study are from tiger salamander, and the expressed opsins used for biochemical experiments are those from salamander and human. Photoreceptor cells are generally identified by cell morphology and the type of opsin it contains that can be further complicated by the findings that some cone cells have multiple opsins (12, 13). Recently genetic analysis has determined that opsins fall into five classes (reviewed in Refs. 14 and 15). We have studied opsins falling into four of these classes and use common color-derived names for the opsins and photoreceptor cells. The classic rod cells used for scotopic vision contain rhodopsin, the visual pigment for the rod opsin (RH1 opsin) and appeared red and thus have been designated as red rods. Some species such as salamanders have an additional rod cell whose photosensitivity is blue-shifted from that of the red rod and thus designated as green rods. In the tiger salamander, the green rods contain the identical opsin (SWS2 opsin) found in blue cones (16). The human blue cones contain an opsin from a different class (SWS1 opsin), which is homologous to the salamander UV cone opsin. The human red and green and salamander red cone opsins all belong to the same class of opsins (M/LWS opsins). Absorption properties of visual pigments are further modulated in some animals including the tiger salamander by use of 11-cis-retinal with an additional double bond (3,4-dehydro or A2 11-cis-retinal) resulting in red-shifted absorbance from pigments containing 11-cis-retinal (A1 11-cis-retinal).We show here that 11-cis-retinol is not an agonist to cone opsins and does not itself generate a light-sensitive opsin. We further show using microspectrophotometry that both red and blue salamander cone cells regenerate visual pigments from 11-cis-retinol, whereas pigments could not be regenerated with 11-cis-retinol in bleached salamander red and green rods even though the latter contains the same opsin as the salamander blue cone. Thus, rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type, and these mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.     

Specific photoisomerization of retinal in squid rhodopsin and metarhodopsin

The composition of retinal isomers in the photosteady-state mixtures formed from squid rhodopsin and metarhodopsin was determined by high-pressure liquid chromatography. A large amount of 9-cis-retinal was obtained at liquid N₂ temperature when rhodopsin was irradiated with orange light, but only small quantities of 9-cis-retinal were obtained at 15°C. Scarcely any 9-cis-retinal was produced from metarhodopsin by irradiation at liquid N₂ temperature. A large quantity of 7-cis-retinal was found in the photoproduct of rhodopsin irradiated at solid carbon dioxide temperature, but not at 15°C and liquid N₂ temperature. 7-cis-Retinal was not produced from metarhodopsin at any temperatures. These results indicate that the photoisomerization of retinal is regulated by the structure of the retinal-binding site of this protein. The formation of 9-cis- and 7-cis-retinals is forbidden in the metarhodopsin protein.     

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

The metabolism of 13-cis-[11-³H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.     

Four acylated flavonol glycosides from leaves of Strychnos variabilis

Four new acylated flavonol glycosides have been isolated and identified from the leaves of Strychnos variabilis: quercetin 3-(4″-trans-p-coumaroyl)robinobioside-7-glucoside (variabiloside A) and its cis derivative (variabiloside B), kaempferol 3-(4″-trans-p-coumaroyl)robinobioside-7-glucoside (variabiloside C) and its cis derivative (variabiloside D).     

Cyclisation of 1-trans-1′-norsqualene-2,3-epoxide and 1-cis-1′-norsqualene-2,3-epoxide by a cell free system of corn embryos

Squalene-2,3-epoxide-cycloartenol cyclase and cycloeucalenol-obtusifoliol isomerase activities were found in microsomal fractions of corn (Zea mays) embryos. Squalene-2,3-epoxide, 1-trans-1′-norsqualene-2,3-epoxide and 1-cis-1′-norsqualene-2,3-epoxide were incubated. Squalene-2,3-epoxide was cyclized giving only cycloartenol, whereas 1-trans-1′-norsqualene-2,3-epoxide gave 31-norcycloartenol and 31-norlanosterol with a reduced yield, 1-cis-1′-norsqualene-2,3-epoxide was not significantly cyclized.     

Cis-4-hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid from Calliandra

cis-4-Hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid were isolated from leaves of Calliandra pittieri. A system for resolving the eight imino acids isolated from Calliandra is described.     

Microbiological Hydroxylation of Cinerone to Cinerolone

Cinerone [2-(2′-cis-butenyl)-3-methyl-2-cyclopenten-1-one] is hydroxylated to cinerolone [2-(2′-cis-butenyl)-3-methyl-4-hydroxy-2-cyclopenten-1-one] by a number of streptomycetes, bacteria, and fungi. Aspergillus niger ATCC 9,142 and Streptomyces aureofaciens ATCC 10,762 were found to be the most effective hydroxylators. The optical activity of the product was found to range from [α]_D²⁵ 0° to -8.6°, depending on the organism and conditions of culture. Two additional hydroxylated products of cinerone have been isolated and identified as 2-n-butyl-4-hydroxy-3-methyl-2-cyclopenten-1-one and 2-(2′-cis-butenyl-4′-hydroxy)-3-methyl-2-cyclopenten-1-one, respectively.     

Epoxyoctadecadienoic acids from Crepis conyzaefolia seed oil

The seed oil of Crepis conyzaefolia (Gouan) Dalle Torre contains previously unidentified (±)-cis-12,13-epoxyoctadeca-trans-6-cis-9-dienoic (14%) and cis-12,13-epoxyoctadeca-cis-6-cis-9-dienoic (2%) acids and the more common vernolic [(±)-12,13-epoxyoctadec-cis-9-enoic] (32%) acid.     

Natural and synthetic diastereoisomeric (?)-3′,4′,7-trihydroxyflavan-3,4-diols

1. Rhodesian copalwood (Guibourtia coleosperma) contains three diastereo-isomeric leuco-fisetinidins. These consist of the (−)-2,3-cis–3,4-cis (2R,3R,4R) and (−)-2,3-cis–3,4-trans (2R,3R,4S) 3′,4′,7-trihydroxyflavan-3,4-diols, and the third was shown to be a 2,3-trans–3,4-cis isomer by means of paper ionophoresis. 2. There occurrence in similar proportions as tannin precursors also in the tropical hardwoods G. tessmannii and G. demeusii implies a close taxonomic relationship between these, and with G. coleosperma. 3. Epimerization of the natural (−)-3′,4′,7- trihydroxy-2,3-trans-flavan-3,4-trans-diol affords a mixture from which the (−)-2,3-cis–3,4-cis isomer was separated readily, but the (−)-2,3-trans–3,4-cis isomer was obtained with difficulty. These were formed by epimerization of the (−)-2,3-trans–3,4-trans isomer at C-2 and C-4, and at C-4, respectively.     

Diastereoisomeric leucoanthocyanidins from the heartwood of acacia melanoxylon

The isolation of two pairs of diastereoisomeric leucoanthocyanidins, namely (2R,3R,4R)-2,3-cis-3,4-cis-3,3′,4,4′,7,8-hexahydroxyflavan or melacacidin, (2R,3R,4S)-2,3-cis-3,4-trans-3,3′,4,4′,7,8-hexahydroxyflavan or isomelacacidin and(2R,3R,4R)-2,3-cis-3,4-cis-4-ethoxy-3,3′,4′,7,8-pentahydroxyflavan or 4-O-ethylmelacacidin, (2R,3R,4S)-2,3-cis-3,4-trans-4-ethoxy-3,3′,4′,7,8-pentahydroxyflavan or 4-O-ethylisomelacacidin is described. 4-O-Ethylmelacacidin is a new compound and all four leucoanthocyanidins are natural constituents of the heartwood of Acacia melanoxylon. Melacacinidin is the name proposed for the anthocyanidin 3,3′,4′,7,8-pentahydroxyflavylium and leucomelacacinidins for the corresponding leucoanthocyanidins. Quinone-methide formation is proposed to account for the difference in reactivity between the diastereoisomers.     

A Ligand Channel through the G Protein Coupled Receptor Opsin

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 Å and is between 11.6 and 3.2 Å wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal β-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90° elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through B.