The solubilization of carotenogenic enzymes of Phycomyces blakesleeanus

The effect of nine ionic and nine non-ionic detergents, over a 0.3–3.0% (w/v) concentration range, on the activity of the enzymes which convert [2-¹⁴C]mevalonic acid into phytoene (7,8,11,12,7′,8′,11′,12′-ψ,ψ-carotene) and β-carotene (β,β-carotene) has been investigated with cell extracts of the C115 carS42 mad-107(?) (β-carotene-accumulating) strain of Phycomyces blakesleeanus. The enzymes catalyzing the conversion of mevalonic acid into phytoene in the C115 and the C5 carB10(?) (phytoene-accumulating) strains of Phycomyces could be released from membranes with high molarity Tris-HCl buffer, but the other carotenogenic enzymes required solubilization with detergents. Enzymic activity was retained with only two ionic detergents (Zwittergents 3–8 and 3–10), whilst Tweens 40 and 60 were the least inhibitory of the non-ionic surfactants. Both Tween 60 and Zwittergent 3–08 solubilized almost 50% of the enzymic activities for the conversion of phytoene to β-carotene, but the former preparation was significantly more stable on storage at ?70°C.     

β-Carotene biosynthesis by extracts of the C115 mutant of Phycomyces blakesleeanus

A cell extract of the yellow C115 car-42 mad-107(?) mutant of Phycomyces blakesleeanus, capable of converting MVA-[2-¹⁴C] into isoprenoids, was used to investigate the formation of β-carotene. The incorporation of radioactivity into β-carotene was reduced by the addition of unlabelled carotenes, solubilised using detergent, to the incubation mixtures. On reisolation of these carotenes after anaerobic incubations, they were found to carry radioactivity. The relative efficiencies of these carotenes as trapping agents are discussed in relation to the pathways of carotene cyclisation and to the apparent operation of a system for the negative feedback control of carotene biosynthesis.     

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

Abscisic acid is considered an apocarotenoid formed by cleavage of a C-40 precursor and subsequent oxidation of xanthoxin and abscisic aldehyde. Confirmation of this reaction sequence is still awaited, and might best be achieved using a cell-free system capable of both carotenoid and abscisic acid biosynthesis. An abscisic acid biosynthesizing cell-free system, prepared from flavedo of mature orange fruits, was used to demonstrate conversion of farnesyl pyrophosphate, geranylgeranyl pyrophosphate and all-trans-β-carotene into a range of β,β-xanthophylls, xanthoxin, xanthoxin acid, 1′,4′-trans-abscisic acid diol and abscisic acid. Identification of product carotenoids was achieved by high-performance liquid chromatography and on-line spectral analysis of individual components together with co-chromatography. Putative C-15 intermediates and product abscisic acid were identified by combined capillary gas chroma-tography-mass spectrometry. Kinetic studies revealed that β-carotene, formed from either famesyl pyrophosphate or geranylgeranyl pyrophosphate, reached a maximum within 30 min of initiation of the reaction. Thereafter, β-carotene levels declined exponentially. Catabolism of substrate β-carotene into xanthophylls, putative abscisic acid precursors and product abscisic acid was restricted to the all-trans-isomer. However, when a combination of all-trans- and 9-cis-β-carotene in the ratio 1:1 was used as substrate, formation of abscisic acid and related metabolites was enhanced. Biosyn-thetically prepared [¹⁴C]-all-trans-violaxanthin, [¹⁴C]-all-trans-neoxanthin and [¹⁴C]-9′-cis-neoxanthin were used as substrates to confirm the metabolic interrelationship between carotenoids and abscisic acid. The results are consistent with 9′-cis-neoxan-thin being the immediate carotenoid precursor to ABA, which is oxidatively cleaved to produce xanthoxin. Formation of abscisic aldehyde was not observed. Rather, xanthoxin appeared to be converted to abscisic acid via xanthoxin acid and 1′,4′-trans-abscisic acid diol. An alternative pathway for abscisic acid biosynthesis is therefore proposed.     

Megastigmane and 7,9′-dinorlignan glycosides from the tubers of Stephania kaweesakii

Two isomers of megastigmane glycosides, (6R, 9S)-blumenol C 9-O-gentibioside (2) and (6S, 9S)-blumenol C 9-O-gentiobioside (3), and a new 7,9′-dinorlignan glycoside, stepdonorlignoside (4) were isolated from the tubers of Stephania kaweesakii. The structure determinations were considered based on the physical data and spectroscopic evidence. The absolute configurations of two megastigmanes were determined for the first time. Additionally, ten known compounds were isolated: (6R, 9S)-blumenol C 9-O-β-D-glucopyranoside, (+)-isolariciresinol 3a-O-β-D-glucopyranoside, salidroside, N-trans-caffeoyltyramine, (R)-isococlaurine, (R)-isococlaurine 4′-O-β-glucopyranoside, (−)-oblongine, (+)-magnocurarine, fordianoside, and (−)-cyclanoline.     

Conversion of a 24β-ethyl-25-methylene intermediate into poriferasterol by Trebouxia species

Clerosterol-[26-¹⁴C], a 24β-ethyl-25-methylene sterol [(24S)-24-ethylcholesta-5,25-dien-3β-ol], was incorporated into clionasterol and poriferasterol by cultures of the green algae Trebouxia sp. 213/3 and Trebouxia sp. 219/2. Degradation of the labelled poriferasterol showed that the ¹⁴C retained its identity and was not incorporated as a result of metabolism of the clerosterol-[26-¹⁴C] and randomisation of label. These results are consistent with the proposed production, and subsequent reduction, of a 24β-ethyl-25-methylene intermediate in 24β-ethyl sterol biosynthesis in algae of the order Chlorococcales.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Cyclization and hydroxylation stereochemistry in the biosynthesis of gibberellic acid

In Gibberella fujikuroi cultures, ent-[3β-³H,17-¹⁴C]kaurene is converted to gibberellic acid with retention of the tritium label at the 3α-position. This evidence for the stereochemistry of 3-hydroxylation also permits the stereochemistry of the ‘proton-initiated’ cyclization step in gibberellic acid biosynthesis to be deduced.     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Alkenylresorcinols and cytotoxic activity of the constituents isolated from Labisia pumila

Phytochemical investigation on the leaves of Labisia pumila (Myrsinaceae), an important medicinal herb in Malaysia, has led to the isolation of 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol (1), labisiaquinone A (2) and labisiaquinone B (3). Along with these, 16 known compounds including 1-O-methyl-6-acetoxy-5-pentadecylresorcinol (4), 5-(pentadec-10Z-enyl)resorcinol (5), 5-(pentadecyl)resorcinol (6), (−)-loliolide (7), stigmasterol (8), 4-hydroxyphenylethylamine (9), 3,4,5-trihydroxybenzoic acid (10), 3,4-dihydroxybenzoic acid (11), (+)-catechin (12), (−)-epicatechin (13), kaempferol-3-O-α-rhamnopyranosyl-7-O-β-glycopyranoside (14), kaempferol-4′-O-β-glycopyranoside (15), quercetin-3-O-α-rhamnopyranoside (16), kaempferol-3-O-α-rhamnopyranoside (17), (9Z,12Z)-octadeca-9,12-dienoic acid (18) and stigmasterol-3-O-β-glycopyranoside (19) were also isolated. The structures of these compounds were established on the basis of 1D and 2D NMR spectroscopy techniques (¹H, ¹³C, COSY, HSQC, NOESY and HMBC experiments), mass spectrometry and chemical derivatization. Among the constituents tested 1 and 4 exhibited strongest cytotoxic activity against the PC3, HCT116 and MCF-7 cell lines (IC₅₀ values ⩽10 μM), and they showed selectivity towards the first two-cell lines relative to the last one.     

Stereochemistry of phytoene

Samples of phytoene (7,8,11,12,7′,8′,11′,12′-octahydro-ψ,ψ-carotene) isolated from higher plant sources, from Neurospora crassa and strains of Phycomyces blakesleeanus and from diphenylamine-inhibited cultures of Rhodospirillum rubrum have been examined by a number of physical methods. All the organisms accumulate predominantly 15-cis phytoene while only traces of all-trans phytoene are normally present. A comparison with synthetic model compounds has shown that the predominant isomer has a trans,cis,trans triene chromophore.     

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Methoxyspheroidene and methoxyspheroidenone,two carotenoids from Rhodopseudomonas spheroides

Two new carotenoids isolated from Rhodopseudomonas spheroides (Rhodospirillaceae) have been identified as methoxyspheroidene (1,1′-dimethoxy-3,4-didehydro-1,2,1′,2′,7$\text{,}\text{?}$8′-hexahydro-ψ,ψ-carotene) obtained from anaerobic cultures and methoxyspheroidenone (1,1′-dimethoxy-3,4-didehydro-1,2,1′,2′,7′,8′-hexahydro-ψ,ψ-caroten-2-one) recovered from aerobic cultures.     

The biosynthesis of the thiazole moiety of thiamine in Salmonella typhimurium

The mechanism of biosynthesis of 4-methyl-5-β-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various ¹⁴C-labeled precursors was determined. We found that [Me-¹⁴C]methionine, [2-¹⁴C]methionine, [U-¹⁴C]alanine, and [2-¹⁴C]glycine were not incorporated whereas [2-¹⁴C]-tyrosine was incorporated. Degradation of the 4-methyl-5-β-hydroxyethyl thiazole obtained after [2-¹⁴C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-β-hydroxyethyl thiazole biosynthesis.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

Covalent anthocyanin–flavonol complexes from the violet-blue flowers of Allium ‘Blue Perfume’

Three covalent anthocyanin–flavonol complexes (pigments 1–3) were extracted from the violet-blue flower of Allium ‘Blue Perfume’ with 5% acetic acid-MeOH solution, in which pigment 1 was the dominant pigment. These three pigments are based on delphinidin 3-glucoside as their deacylanthocyanin and were acylated with malonyl kaempferol 3-sophoroside-7-glucosiduronic acid or malonyl-kaempferol 3-p-coumaroyl-tetraglycoside-7-glucosiduronic acid in addition to acylation with acetic acid.By spectroscopic and chemical methods, the structures of these three pigments 1–3 were determined to be: pigment 1, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(trans-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate; pigment 2, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-β-glucopyranosyl^III)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))); and pigment 3, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(cis-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate.The structure of pigment 2 was analogous to that of a covalent anthocyanin–flavonol complex isolated from Allium schoenoprasum where delphinidin was observed in place of cyanidin. The three covalent anthocyanin–flavonol complexes (pigment 1–3) had a stable violet-blue color with three characteristic absorption maxima at 540, 547 and 618 nm in pH 5–6 buffer solution. From circular dichroism measurement of pigment 1 in the pH 6.0 buffer solution, cotton effects were observed at 533 (+), 604 (−) and 638 (−) nm. Based on these results, these covalent anthocyanin–flavonol complexes were presumed to maintain a stable intramolecular association between delphinidin and kaempferol units closely related to that observed between anthocyanin and hydroxycinnamic acid residues in polyacylated anthocyanins. Additionally, an acylated kaempferol glycoside (pigment 4) was isolated from the same flower extract, and its structure was determined to be kaempferol 3-O-sophoroside-7-O-(3-O-(malonyl)-β-glucopyranosiduronic acid).     

Biosynthesis of carotenoids by Phycomyces blakesleeanus mutants in the presence of nitrogenous heterocyclic compounds

Pyridine, imidazole and some of their derivatives stimulate lycopene and γ-carotene synthesis-simultaneously inhibiting β-carotene formation in Phycomyces blakesleeanus Strain C115. Isonicotinoly-hydrazine has a toxic effect on Strains C9 and C115 and 1-methylimidazole on Strain C115 in the concentrations of 1 g/l. Compounds which cause an accumulation of lycopene and γ-carotene usually cause an increase in phytoene synthesis and the disappearance of β-zeacarotene. The effect of succinimide, 4-hydroxypyridine, and isonicotinoylhydrazine on Strain C9 has also been studied. When β-picoline and 2-methylimidazole treated C115 mycelia were washed and resuspended in phosphate buffer at pH 5·6 β-zeacarotene reappeared and β-carotene increased with the simultaneous decrease in lycopene and γ-carotene. The sum of β-carotene, γ-carotene up to 3days of resuspension was almost equal to the total of these at zero time. These results show that the inhibitory action of these compounds is on the enzymes responsible for cyclization of acyclic carotenes. This inhibition varies with the nature of the substituent on the heterocyclic ring and pyridine derivatives having pK_a values of 6 ± 1 show the greatest degree of inhibition.     

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ?¹²-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ?¹⁵-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ?⁹-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.     

Iridoid and phenylethanoid glycosides from Heterophragma sulfureum

An unusual iridoid diglycoside (specioside 6′-O-α-d-galactopyranoside) and a new phenylethanoid triglycoside (heterophragmoside) were isolated from the leaves and branches of Heterophragma sulfureum together with specioside, verminoside, 6-trans-feruloylcatapol, stereospermoside, (−)-lyoniresinol 3α-O-β-d-glucopyranoside, (+)-lyoniresinol 3α-O-β-d-glucopyranoside, (−)-5′-methoxyisolariciresinol 3α-O-β-d-glucopyranoside, (+)-5′-methoxyisolariciresinol 3α-O-β-d-glucopyranoside, and dehydroconiferyl 4-O-β-d-glucopyranoside. The structural elucidations were based on analyses of chemical and spectroscopic data.     

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.     

Enzymatic synthesis of carotenes by cell-free preparations of fruit of several genetic selections of tomatoes

Several mutants of tomatoes are known in which the carotene content of the fruit is markedly altered qualitatively and quantitatively from that found in the standard red tomato variety. These selections are: rr (yellow flesh, low carotene); tt (tangerine, orange, proneurosporene and prolycopene); at at (apricot, low in acyclic carotenes); og^c og^c (crimson, high in lycopene); Verkerk 377-2αα (probably identical to vircscent orange vo vo, high in ζ-carotene); B B (H_i-β, high in β-carotene), and Del Del (Hi-δ, high in δ-carotenc). Studies of carotene synthesis from [1-¹⁴C]isopentenyl pyrophosphate, [¹⁴C]phytoene, and [¹⁴C]lycopcne by soluble enzyme systems obtained from fruits of these selections have shown unexpected enzyme activities. All selections evidence activity for the synthesis of phytoene. All mutants have also been found to contain an enzyme system for the synthesis of β-carotenefrom lycopene. Three of the selections analyzed (rr, at at, and og^c og^c) also contain an enzyme system for the conversion of lycopene to α-carotene and the variants rr and tt contain an enzyme for the synthesis of poly-cis-carotencs from isopentenyl pyrophosphate and phytocne.The reasons for the discrepancies that are observed between carotene composition of fruit of field-grown tomato selections and enzyme activities for carotene synthesis by cell-free preparations obtained from these fruits are not presently known. It is obvious, however, that either inhibitors are present, cofactors are missing, or there are permeability barriers to substrate or cofactor transport into plastids of selections in which enzyme activities are not expressed in field-grown fruit. Further investigations will be required for clarification of this problem.