28-Norcastasterone is biosynthesized from castasterone

Metabolic experiments with deuterium-labeled castasterone in seedlings of Arabidopsis thaliana, Oryza saliva and Lycopersicon esculentum, and cultured cells of Catharanthus roseus were performed, and the metabolites were analyzed by GC-MS. In all the plant species examined, [2H3]28-norcastasterone was identified as a metabolite of [26,28-2H6]castasterone, indicating that castasterone is the biosynthetic origin of 28-norcastasterone. Moreover, the natural occurrence of 28-norcastasterone and 28-nortyphasterol in seedlings of A. thaliana has been demonstrated. This is the first report of the natural occurrence of 28-nortyphasterol in plants.     

Biosynthetic pathways of brassinolide in Arabidopsis

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-dehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6alpha-hydroxycastasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone --> 3-dehydroteasterone --> typhasterol --> castasterone --> BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.     

Biosynthesis of cholestanol in higher plants

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.     

Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L

GC-MS analysis revealed that primary roots of maize contain 6-deoxocathasterone, 6-deoxoteasterone and 6-deoxotyphasterol. These brassinosteroids, and the previously identified campesterol, campestanol, 6-deoxocastasterone and castasterone, in the roots are members of a biosynthetic pathway to castasterone, namely the late C-6 oxidation pathway, suggesting that its biosynthetic pathway is operative in the roots. To verify this, a cell-free enzyme extract was prepared from maize roots, and enzymatic conversions from campesterol to castasterone through the aforementioned sterols and brassinosteroids were examined. The presence for the biosynthetic sequences, campesterol-->24-methylcholest-4-en-3beta-ol-->24-methylcholest-4-en-3-one-->24-methylcholest-5 alpha-cholestan-3-one-->campestanol and 6-deoxoteasterone-->6-deoxo-3-dehydroteasterone-->6-deoxotyphasterol-->6-deoxocastasterone-->castasterone were demonstrated. These results indicate that maize roots contain a complete set of enzymes involved in the late C-6 oxidation pathway, thereby demonstrating that endogenous brassinosteroids are biosynthesized in the roots.     

Synthesis and biological activity of 26-norbrassinolide, 26-norcastasterone and 26-nor-6-deoxocastasterone

26-Norbrassinolide, identified as a metabolite of brassinolide in cultured cells of the liverwort, Marchantia polymorpha, as well as 26-norcastasterone and 26-nor-6-deoxocastasterone were synthesized. Synthesis of these new brassinosteroids was conducted by employing the orthoester Claisen rearrangement and asymmetric dihydroxylation as key reactions. The modified rice lamina inclination test indicated that these three 26-norbrassinosteroids were less active than their corresponding C28 brassinosteroids. Growth-promoting activities were also examined by using the brassinosteroid-deficient, dwarf mutant lkb of garden pea (Pisum sativum L.). In this assay, 26-norbrassinolide was as effective as brassinolide and 26-norcastasterone was more effective than castasterone although 26-nor-6-deoxocastasterone was much less effective than 6-deoxocastasterone. Therefore, removal of C-26 of brassinosteroids does not necessarily reduce the biological activity. The role of C-26 removal in Marchantia cells remains unclear.     

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre. are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.     

Biosynthesis and metabolism of brassinosteroids

Natural brassinosteroids so far identified from various plant species include biosynthetic congeners of brassinolide, such as cathasterone, teasterone, 3-dehydroteasterone, typhasterol and castasterone as well as another series of 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone, 6-deoxotyphasterol and 6-deoxocastasterone. Using cell culture system of Catharanthus roseus , the outlines of biosynthetic pathways of brassinolide, via plant sterol of campesterol, have now been demonstrated. There are two pathways, named early C6-oxidation pathway and late C6-oxidation pathway, both of which would be operating in wide varieties of plants. Metabolic studies with various plant systems revealed multiple paths of metabolism such as hydroxylation, epimerization, side chain cleavage, reduction and conjugation with glucose and fatty acids. Recent progress of biosynthesis and metabolism of brassinosteroids is described.     

The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.     

Identification and quantification of brassinolide-related steroids in the insect gall and healthy tissues of the chestnut plant

Castasterone and 6-deoxocastasterone were identified by GC/MS and/or selected ion monitoring not only in the insect gall of chestnut but also in the healthy tissues, including the shoot, leaf and flower bud. In addition, the gall was found to contain a small amount of brassinolide. Brassinone, which had been reported to be present in the gall, could not be detected in these tissues. The contents of castasterone, 6-deoxocastasterone and brassinolide, which were determined by using selected ion monitoring, are discussed.     

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. It has been proposed that BRs are synthesized via two parallel pathways, the early and late C-6 oxidation pathways according to the C-6 oxidation status. The tomato (Lycopersicon esculentum) Dwarf gene encodes a cytochrome P450 that has been shown to catalyze the C-6 oxidation of 6-deoxocastasterone to castasterone. We isolated an Arabidopsis ortholog (AtBR6ox gene) of the tomato Dwarf gene. The encoded polypeptide has characteristics of P450s and is classified into the CYP85 family. The AtBR6ox and tomato Dwarf gene were expressed in yeast and the ability of the transformed yeast cells to metabolize 6-deoxo-BRs was tested. Metabolites were analyzed by gas chromatography-mass spectrometry. Both enzymes catalyze multiple steps in BR biosynthesis: 6-deoxoteasterone to teasterone, 3-dehydro-6-deoxoteasterone to 3-dehydroteasterone, 6-deoxotyphasterol to typhasterol, and 6-deoxocastasterone to castasterone. Our results indicate that the AtBR6ox gene and the tomato Dwarf gene encode steroid-6-oxidases and that these enzymes have a broad substrate specificity. This suggests that the BR biosynthetic pathway consists of a metabolic grid rather than two separate parallel pathways.     

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea

Endogenous brassinosteroids (BRs) in the dwarf mutants lka and lkb of garden pea (Pisum sativum L.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated internal standards. In young shoots of the lkb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone normalized internode growth of lkb seedlings. These findings indicate that the lkb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of lka plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The lka seedlings were approximately 100 times less responsive to brassinolide than the lkb mutant, and application of castasterone had only a marginal effect on lka internode growth, suggesting that the lka lesion results in impaired sensitivity to BR.     

Isolation and characterization of brassinosteroids from algal cultures of Chlorella vulgaris Beijerinck (Trebouxiophyceae)

The brassinosteroids (BRs) occur ubiquitously in the plant kingdom. The occurrence of BRs has been demonstrated in almost every part of higher plants, such as pollen, flower buds, fruits, seeds, vascular cambium, leaves, shoots and roots. In this study, BRs were isolated and identified in the culture of wild-type Chlorella vulgaris. Seven BRs, including teasterone, typhasterol, 6-deoxoteasterone, 6-deoxotyphasterol, 6-deoxocastasterone, castasterone and brassinolide, were identified by GC–MS. All compounds belong to the BR biosynthetic pathway. The results suggest that early and late C6 oxidation pathways are operating in C. vulgaris. This study represents the first isolation of BRs from C. vulgaris cultures.     

Metabolism of gibberellins A1 and A3 in fruits and shoots of Prunus avium

Isotope-labelled GA metabolites were identified by GC--MS, following HPLC fractionation of extracts derived from fruits or shoots, that had been incubated with [2H]- and [3H]- GA1 or [2H]- and [3H]- GA3. GA1 (1) was converted into GA8 (10) by developing fruits and vegetative shoots of sweet cherry (Prunus avium cv. 'Stella'), while GA3 (4) was converted into GA3-isolactone (17). Other metabolites of each GA were detected but were not identified unequivocally. These included a metabolite of GA1 (1) in fruitlets that was more polar (by reverse phase HPLC) than GA8 (10) and a metabolite of similar polarity to GA87 (6), was obtained after incubating fruitlets with GA3 (4). However, no evidence was obtained to suggest that GA87 (6) was a metabolite of GA3 (4) or that GA85 (2) was a metabolite of GA1 (1) in these tissues, under the conditions used. The pattern of metabolites obtained from vegetative tissues was similar to that from fruitlets. However, the results suggested that GA1 (1) and GA3 (4) were metabolised at a greater rate in shoots from mature trees than in shoots from seedlings, and that GA1 (1) was metabolised more rapidly than GA3 (4) in juvenile and mature shoots. We conclude from these observations that GA3 (4) is not a precursor of GA87 (6) and GA32 (5), also, that GA1 (1) is not a precursor of GA85 (2) and GA86 (3) in developing fruits or in vegetative shoots of sweet cherry.     

Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol

Escherichia coli were grown on 14.3% uniformly 13C-labeled glucose as the sole carbon source and challenged anaerobically with 90% 13C-labeled formaldehyde. The major multiply labeled metabolites were identified by 13C NMR spectroscopy to be glycerol and 1,2-propanediol, and a minor metabolite was shown to be 1,3-propanediol. In each case, formaldehyde is incorporated only into the C1 position. A novel form of 13C NMR isotope dilution analysis of the major products reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of the glycerol C1 is derived from bacterial sources. Glycerokinase converted the metabolite [1-13C]glycerol to equal amounts of [3-13C]glycerol 3-phosphate and [1-13C]glycerol 3-phosphate, demonstrating that the metabolite is racemic. When [13C]formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium incorporation levels are different for each metabolite, indicating that the biosynthetic pathways probably diverge early.     

Purification and Separation of Eight Steroidal Plant-growth Regulators from Dolichos lablab Seed

Purification and separation of eight steroidal plant-growth regulators, dolicholide, dolichosterone, homodolicholide, homodolichosterone, brassinolide, castasterone, 6-deoxocastasterone and 6-deoxodolichosterone, from immature seed of Dolichos lablab were accomplished using various chromatographic techniques. The reversed-phase HPLC of a number of steroidal plant growth regulators was also studied.     

N6-Trimethyl-lysine metabolism. 3-Hydroxy-N6-trimethyl-lysine and carnitine biosynthesis.

Rats injected with N6-[Me-3H]trimethyl-lysine excrete in the urine five radioactively labelled metabolites. Two of these identified metabolites are carnitine and 4-trimethylammoniobutyrate. A third metabolite, identified as 5-trimethylammoniopentanoate, is not an intermediate in the biosynthesis of carnitine; the fourth and major metabolite, N2-acetyl-N6-trimethyl-lysine, is not a precursor of carnitine. The remaining metabolite (3-hydroxy-N6-trimethyl-lysine) is converted into trimethylammoniobutyrate and carnitine by rat liver slices and into trimethylammoniobutyrate by rat kidney slices. In rat liver and kidney-slice experiments, radioactivity from DL-N6-trimethyl-[1-14C]lysine and DL-N6-trimethyl-[2-14C]lysine was incorporated into N2-acetyl-N6-trimethyl-lysine and 3-hydroxy-N6-trimethyl-lysine, but not into trimethylammoniobutyrate or carnitine. A procedure was devised to purify milligram quantities of 3-hydroxy-N6-trimethyl-lysine from the urine of rats injected chronically with N6-trimethyl-lysine (100 mg/kg body wt. per day). The structure of 3-hydroxy-N6-trimethyl-lysine was confirmed chemically and by nuclear-magnetic-resonance spectrometry [Novak, Swift & Hoppel (1980) Biochem. J. 188, 521--527]. The sequence for carnitine biosynthesis in liver is: N6-trimethyl-lysine leads to 3-hydryxy-N6-trimethyl-lysine leads to leads to 4-trimethylammoniobutyrate leads to carnitine.     

The metabolism of aldosterone and 3 alpha, 5 beta-tetrahydroaldosterone in the rabbit

Analysis of urinary metabolites of [1, 2-3H]-aldosterone and [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone was performed in male rabbits. The preliminary separation of urinary metabolites was carried out by submitting these metabolites to countercurrent distribution. Further separation of each fraction thus obtained was achieved by means of DEAE-Sephadex A-25 column chromatography. The separated peak was then hydrolyzed with the enzyme and the free steroid released was identified on the basis of the mobilities of the steroid and its derivatives on paper chromatography. After the injection of [1, 2-3H]-aldosterone, a major urinary metabolite was characterized as monosulphate of 3 alpha, 5 beta-tetrahydroaldosterone. In addition, a small amount of the monoglucosiduronate fraction was found in the urine. 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were detected as aglycones in this fraction. After the injection of [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone, a similar pattern of urinary radiometabolites was observed. The close similarity between the profile of urinary metabolites of [1, 2-3H]-aldosterone and that of [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone suggests that the conversion of aldosterone to 3 alpha, 5 beta-tetrahydroaldosterone is needed before the conjugation processes take place.     

The fate of corticosterone and 11-deoxycorticosterone in C57BL/6 and BALB/c strains of mice: distribution and oxidative metabolism

The distribution kinetics and oxidative metabolism of [4-C14] corticosterone (B) and 11-deoxy-[1,2-3H] corticosterone (DOC) were compared in C57BL/6 (B6) and BALB/c (C) mice. Statistically important differences in the distribution of [14C]B and [3H]DOC occurred that were independent of strain, while other differences were strain dependent. Intestinal excretion of metabolites of B and DOC was greater in B6 mice than in C mice, and kidney excretion was greater in C mice than B6 mice. In both C and B6 mice, 3H was cleared from liver faster than 14C, with no strain differences. DOC metabolite levels exceeded B metabolite levels in small intestine and gall bladder of both strains. In most other organs, B metabolites exceeded DOC metabolites. Time average strain differences in accumulation of B and its metabolites favoring B6 were found in pancreas, brain, lung, heart, muscles, adrenals, spleen, mesentery and small intestine. Except for the organs of excretion, no strain differences were found for [3H]DOC metabolites. Sixty minutes after steroid administration, 45% of B metabolites and a third of DOC metabolites were 20-hydroxy-21-oic acids. In the intestine, accumulation of acids derived from either B or DOC was greater for B6 than C strain mice, reflecting the greater proportion of total steroid excreted in the B6 strain.     

Bacterial Transformations of Naphthothiophenes

Naphthothiophenes are minor components of fossil fuels, and they can enter the environment from oil spills. Naphtho[2,1-b]thiophene, naphtho[2,3-b]thiophene, and 1-methylnaphtho[2,1-b]thiophene were synthesized and used in biodegradation studies with 1-methylnaphthalene (1-MN)-degrading Pseudomonas strains W1, F, and BT1. Cultures were incubated with one of the naphthothiophenes with or without 1-MN, acidified, and extracted with CH(inf2)Cl(inf2). The extracts were analyzed by gas chromatography with flame photometric and mass detectors to characterize sulfur-containing metabolites and with an atomic emission detector for quantification. Only strain W1 was able to grow on naphtho[2,1-b]thiophene, but strains F and BT1 cometabolized this compound if 1-MN was present. 1-MN was required by all three strains to metabolize naphtho[2,3-b]thiophene, which was more resistant to biodegradation than the [2,1-b] isomer. Two metabolites of naphtho [2,1-b]thiophene were purified, analyzed by (sup1)H nuclear magnetic resonance spectroscopy, and found to be 4-hydroxybenzothiophene-5-carboxylic acid (metabolite I) and 5-hydroxybenzothiophene-4-carboxylic acid (metabolite II). In cultures of strain W1 grown for 7 days on 52 (mu)mol of naphtho[2,1-b]thiophene, >84% of the substrate was degraded and metabolites I and II accounted for 19 and 9%, respectively, of the original amount of naphtho[2,1-b]thiophene. When 1-MN was present, strain W1 degraded >97% of the naphtho[2,1-b]thiophene and similar amounts of metabolite II were produced, but metabolite I did not accumulate. 1-MN was shown to promote the further degradation of metabolite I, but not of metabolite II, by strain W1. Thus, 1-MN enhanced the biodegradation of naphtho[2,1-b]thiophene. Approximately 70% of the 1-methylnaphtho [2,1-b]thiophene added to cultures of strain W1 with 1-MN was recovered as 4-hydroxy-3-methylbenzothiophene-5-carboxylic acid, the 3-methyl analog of metabolite I. The methyl substitution hindered further metabolism of 3-methyl-metabolite I even in the presence of 1-MN. Cometabolism of naphtho[2,3-b]thiophene yielded two products that were tentatively identified as 5-hydroxybenzothiophene-6-carboxylic and 6-hydroxybenzothiophene-5-carboxylic acids.