Biosynthetic Sequence Research Articles

A non-enzymatic reaction in the late biosynthesis of the decarestrictine family.

In the late biosynthesis of the decarestrictines, inhibitors of the de novo formation of cholesterol isolated from the culture broth of Penicillium simplicissimum (FH-A 6090), a common pentaketide precursor undergoes post-polyketide modifications leading to the different members of the decarestrictine family. Besides subsequent enzymatically catalyzed reactions an unexpected non-enzymatic conversion was found to be the key step in the biosynthetic sequence. Under acidic conditions during fermentation the decarestrictines A1 and A2 (3 and 4) are converted into the main product decarestrictine D (2) and the new decarestrictines N (5) and O (6), whose physico-chemical data are reported. Mechanistic aspects of the non-enzymatic reaction as well as a more detailed picture of the biosynthetic relationships of the decarestrictine family are described. By the application of pH-static fermentations these results were successfully used to manipulate the secondary metabolite pattern of strain FH-A 6090 directing the fermentation process to produce desired members of the decarestrictine family.

Potential inhibitors of trichothecene biosynthesis in Fusarium culmorum: Epoxidation of a trichodiene derivative

A range of semisynthetic trichodiene derivatives was shown to inhibit trichothecene mycotoxin production in cultures of Fusarium culmorum, and to cause accumulation of the biosynthetic intermediate isotrichodiol. 9β,10β-Epoxytrichodiene was particularly effective in inhibiting incorporation of 14C-labelled acetate into trichothecenes, and was also metabolized by the fungus to 9β,10β;12,13-diepoxytrichodiene and 3α-hydroxy-9β,10β;12,13-diepoxytrichodiene. 9β,10β;12,13-Diepoxytrichodiene was similarly transformed to the 3α-hydroxy derivative. This 12,13-epoxidase activity is likely to be the same as that involved in the normal biosynthetic sequence to trichothecenes, the crucial step conferring toxicity to the molecules. Dihydroxycalonectrin (DHC) and 15-deacetylDHC were shown to be efficient precursors of the major F. culmorum trichothecene, 3-acetyldeoxynivalenol (3-AcDON), indicating the last two steps in the biosynthetic sequence to 3-AcDON are 15-deacetylation and then 8-oxidation. 15-DeacetylDHC was characterized as a minor F. culmorum metabolite.

Elimination of photoreceptor (paraflagellar swelling) and photoreception in Euglena gracilis by means of the carotenoid biosynthesis inhibitor nicotine

Nicotine is the chief representative of tobacco alkaloids. It is a commonly used insecticide, and has been reported to be a potent and reversible inhibitor of carotenoid biosynthesis. It inhibits cyclohexylidene ring formation in the biosynthetic sequence leading to β-carotene. This specificity prompted us to investigate the influence of this alkaloid on the photoreceptor of Euglena gracilis in an attempt to confirm our previous microscopic data which indicated that the chromophores present in the photoreceptor could be related to carotenoids. The effects of nicotine at various concentrations on the photoreceptor of Euglena gracilis and its photoreception were investigated using phase-contrast microscopy, fluorescence microscopy, transmission electron microscopy and photobehaviour. Nicotine affects the formation of the photoreceptor, after five cell generations. Photoaccumulation experiments on nicotine-treated Euglena cells clearly show that these cells are unable to see light. These results strongly confirm the presence of a chromophore related to carotenoids in the photoreceptor of Euglena gracilis. On removal of nicotine, cells again acquire their physiological status.

Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product

A protein catalyzing methylation at C-5 and C-15 and decarboxylation of the acetic acid side chain at C-12 on precorrin-6y to yield precorrin-8x was purified to homogeneity from a recombinant strain of Pseudomonas denitrificans. It was sequenced at the N terminus and shown to be encoded by the cobL gene.

Further Studies on the Biosynthesis of Pseudoisoeugenols in Tissue Cultures of Pimpinella anisum

Abstract Tissue cultures of Pimpinella anisum and P. major were used to study the biosynthesis of pseudoisoeugenols. The putative precursors were labelled with 13C with one exception that was labelled with 14C. The incorporation of the label was controlled by 13CNMR and liquid scintil­ lation, respectively. The biosynthetic sequence found was L-phenylalanine, p-cinnamic acid, p-coumaric acid, p-hydroxycoum aric alcohol, p-methoxycoumaric alcohol and anethol. The incorporation rates ranged from 0.5% to 25%.

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine --> ethanolamine --> phosphoethanolamine --> phosphatidylethanolamine. A possible alternative is: serine --> phosphatidylserine --> phosphatidylethanolamine --> ethanolamine --> phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO(2) from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.

β-Glucosides of Aroma Components from Petals of Rosa Species: Assay, Occurrence, and Biosynthetic Implications

Summary Significant proportions of the monoterpenes and of 2-phenylethanol in the petals of sixteen Rosa species and cultivars were bonded as β-D-glucosides. Conjugate-formation was greatest (10 to 43% of total alcohol content) in partially-opened flowerheads in all examples, but the kinetics of labelling of free alcohols and β-glucosides by exogenous [14C]-labelled mevalonate and phenylalanine were complex and revealed no obvious precursor - product relationships. Cell-free extracts of several species and of cell cultures derived therefrom possessed UDP-dependent glycosyl transferase activities towards the free alcohols; no evidence could be adduced for a previously-suggested biosynthetic sequence, whereby the β-glucosides (derived directly from the corresponding pyrophosphates) were obligatory precursors of the free alcohols. During the course of this work, the aglycone-specificities of several β-D-glucohydrolases towards monoterpenyl-β-D-glucosides possessing a wide range of skeletal-types were determined, and enzymatic assays for such glucosides in plant material in general were established.

Synthesis of Methylated Ethanolamine Moieties

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[(3)H(3)C]methionine, l-[(14)CH(3)]methionine, or [1,2-(14)C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.

Conversion of dihydro-O-methylsterigmatocystin to aflatoxin B2 by Aspergillus parasiticus.

Dihydro-O-methylsterigmatocystin (DHOMST) was identified in cultures of Aspergillus parasiticus SRRC 2043, an aflatoxin (AF) non-producer, by comparison of its mass spectrum to that of authentic DHOMST. In addition to DHOMST, SRRC 2043 is known to produce two other chemically related compounds in culture: O-methylsterigmatocystin (OMST) which is an AFB1 precursor and a hydrated-vinyl ether analogue of OMST (HOMST) which is an AFB2 precursor. In the present study, DHOMST also was shown to be an AFB2 precursor by demonstrating that DHOMST is converted to AFB2 by mycelia of an A. parasiticus mutant (SRRC 163) blocked early in the AF biosynthetic pathway; this conversion occurred at an even higher rate than observed for HOMST to AFB2 conversion by the same fungal strain. SRRC 2043 mycelia, although incapable of producing AF's when supplied with OMST, HOMST or DHOMST, were capable of converting HOMST to DHOMST. The results indicated that DHOMST can be placed after HOMST in the biosynthetic sequence which produces AFB2.

Flavonoid 3´-O-Methylation by a Zea mays L. Preparation1

Summary The methylated flavonoids, peonidin and isorhamnetin, found in pigment extracts of maize ( Zea mays L.) tissues are the products of methylation by the S-adenosylmethionine-flavonoid 3´-Omethyltransferase described herein. The transferase is found in seedlings, leaf sheaths, and immature aleurone tissues and requires S-Adenosylmethionine as the methyl donor. S-adenosylhomocysteine inhibited the enzyme 50% at the 27 µM level whereas the same inhibition was observed with sinefungin present at a 1 µM level. The transferase had no apparent metal ion requirement, an optimum temperature of 40 °C and an optimum pH range between 8.0 and 8.5. The enzyme utilized either eriodictyol, a flavanone; luteolin, a flavone; or quercetin a flavonol as the substrate but would not methylate dihydroquercetin, a partially reduced flavonol or quercetin 3-glucoside. This suggests that methylation occurs sometime prior to the glucosylation reaction which is thought to occur near the end of the biosynthetic sequence for the flavonoid compounds in maize. Identification of the biochemical-genetic interrelationships of the transferase with the genes known to be involved in flavonoid biosynthesis in maize remains to be determined.

Characterization of an Escherichia coli rff mutant defective in transfer of N-acetylmannosaminuronic acid (ManNAcA) from UDP-ManNAcA to a lipid-linked intermediate involved in enterobacterial common antigen synthesis

The rff genes of Salmonella typhimurium include structural genes for enzymes involved in the conversion of UDP N-acetyl-D-glucosamine (UDP-GlcNAc) to UDP N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), the donor of ManNAcA residues in enterobacterial common antigen (ECA) synthesis. An rff mutation (rff-726) of Escherichia coli has been described (U. Meier and H. Mayer, J. Bacteriol. 163:756-762, 1985) that abolished ECA synthesis but which did not affect the synthesis of UDP-ManNAcA or any other components of ECA. The nature of the enzymatic defect resulting from the rff-726 lesion was investigated in the present study. The in vitro synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), an early intermediate in ECA synthesis, was demonstrated by using membranes prepared from a mutant of E. coli possessing the rff-726 lesion. However, in vitro synthesis of the next lipid-linked intermediate in the biosynthetic sequence, ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II), was severely impaired. Transduction of wild-type rff genes into the mutant restored the ability to synthesize both lipid II and ECA as determined by in vitro assay and Western blot (immunoblot) analyses done with anti-ECA monoclonal antibody, respectively. Our results are consistent with the conclusion that the rff-726 mutation is located in the structural gene for the transferase that catalyzes the transfer of ManNAcA from UDP-ManNAcA to lipid I.

Biosynthesis and metabolism of sesquiterpenoid phytoalexins and triterpenoids in potato cell suspension cultures

The accumulation and turnover of sesquiterpenoid phytoalexins and the effects of the induction of phytoalexin biosynthesis on triterpenoid synthesis have been studied in potato tuber tissue discs and cell suspension cultures inoculated with sporangia of either an incompatible (Race 4) or compatible (Complex) race of Phytophthora infestans. A comparative study of the incompatible and compatible interaction using Kennebec (R 1) tuber tissue showed the accumulation of rishitin (major component) and lubimin in both interations, though the patterns of accumulation and the observed fungal development were different. Rishitin turnover in both interactions was demonstrated by administration early in the time course of a small dose of [2- 14C]mevalonate though no accumulation of a rishitin metabolite was apparent. The assay of the incorporation over a series of short time periods (2 hr) of either [2- 14C]MVA or [2- 14C]acetate into squalene and sterols suggested that triterpenoid synthesis by tuber discs ( P. infestans is unable to synthesize sterols) was inhibited during the period of phytoalexin accumulation. A similar study with Kennebec-cell suspension cultures showed the accumulation of rishitin, mostly in the growth medium, when the cultures were inoculated with sporangia of P. infestans. Unlike the tuber discs, however, the patterns of accumulation of rishitin were identical with both races of fungus and the zoospores liberated from the sporangia did not germinate and infect the potato cells. A marked loss of rishitin from the inoculated culture occurred after 24 hr, but, as with tuber discs, this loss took place without the accumulation of any rishitin metabolise. As judged by the incorporation over short time periods (2 hr) of 12- 14C]MVA into squalene, sterols and phytoalexins, triterpenoid synthesis was markedly reduced just prior to the onset of phytoalexin accumulation. Potato cv. Majestic (r)-cell suspension cultures inoculated with sporangia of P. infestans Complex race accumulated lubimin, 3-hydroxylubimin, rishitin, phytuberol and phytuberin in the growth medium. The patterns of accumulation of phytoalexins in the presence or absence of a saturating concentration for sterol synthesis of either [2- 14C]MVA (3.3 mM) or 12- 14C]acetate (1 mM) were in agreement with the partial biosynthetic sequence lubimin → 3-hydroxylubimin → rishitin. Triterpenoid synthesis, as measured by the incorporation of the 3.3 mM [2- 14C]MVA and 1 mM [2- 14C]acetate, ceased very abruptly just prior to the start of phytoalexin accumulation. The reason for this appeared to be due to the inhibition or loss of squalene synthetase activity. The presence of 3.3 mM [2- 14C]MVA in the cultures resulted in a large increase in the levels of squalene, squalene 2,3-oxide and cycloartenol in the cells of healthy cultures and of phytoalexins accumulated in the growth medium of the inoculated cultures. This apparent lack of any regulatory control of the incorporation of MVA into the three triterpenoids and into phytoalexins is presumably the principal reason for the need to bring about the rapid and complete inhibition of squalene synthetase activity in the cells of inoculated cultures and, by implication, in the cells responsible for the synthesis of phytoalexins in infected tuber discs.

Microsomal isoflavone 2′- and 3′-hydroxylases from chickpea (Cicer arietinum L.) cell suspensions induced for pterocarpan phytoalexin formation

Microsomal fractions derived from suspension-cultured chickpea (Cicer arietinum L.) cells induced for phytoalexin biosynthesis catalyzed the monohydroxylation of 4′-methoxyisoflavones (biochanin A and formononetin) in the 2′- and 3′-positions. The reactions depended on NADPH and molecular oxygen. Post-microsomal supernatants or microsomes from non-induced cells are without detectable activity in the hydroxylase assay. 4′-Hydroxyisoflavones (genistein and daidzein) were not hydroxylated to any significant extent. The occurrence of these hydroxylases proceeds concomitantly with the accumulation of two pterocarpan phytoalexins, medicarpin and maackiain, by induced cell cultures. The results are discussed with regard to the biosynthetic sequences in the conversion of isoflavones to pterocarpans.

Biosynthesis of artemisinin in Artemisia annua

The isotope ratios ( 3H: 14C) in arteannuin B and artemisinin biosynthesized in Artemisia annua from [4 R- 3H 1,2- 14C]-, [5- 3H 2,2- 14C]- and [2- 3H 2,2- 14C](3 RS)- mevalonate have revealed that two specific 1,2-hydride shifts take place during the oxidation and lactonization of the germacrane skeleton to yield dihydrocostunolide. The gem-methyls of DMAPP retain their identity until the final steps of artemisinin biosynthesis. Arteannuin B is considered to be a late precursor of artemisinin and the following biosynthetic sequence is suggested: farnesylpyrophosphate → germacrane skeleton → dihydrocostunolide → cadinanolide → arteannuin B → artemisinin.

Sterols in marine invertebrates. Part 57. Stereostructure, synthesis, and acid-catalysed isomerization of hebesterol—a biosynthetically significant cyclopropyl-containing marine sterol

Three new cyclopropane-containing sterols hebesterol (11), petrostanol (8), and 23,24-dihydro-5α-calystanol (10) have been isolated from the sponge, Petrosia hebes, together with the principal sterol, petrosterol (7), and 21 known sterols. The structure elucidation of the new trace sterols was accomplished by spectroscopic methods and partial synthesis. Hebesterol (11) and its trans-diastereoisomers (31)–(33) have been synthesized and their acid-catalysed cyclopropane ring-opening studied. Each hebesterol isomer (11), (31)–(33) was correlated with the recently synthesized ficisterol isomers (5), (53), (56), and (58) of known absolute stereochemistry, thus leading to an unambiguous assignment of the absolute stereochemistry of hebesterol. The other products from the acid-catalysed isomerizations were characterized by spectroscopic methods, synthesis, and mechanistic considerations. Two of these products, (23R)-23-ethyldesmosterol (51) and (23S)-23-ethyldesmosterol (59), provided a relay to the absolute stereochemistry of the 23-ethylcholestanols, which had previously been synthesized without assignment of absolute stereochemistry. Hebesterol (11) is the key ‘missing link’ with the predicted stereochemistry in a proposed biosynthetic sequence encompassing the unusual marine sterols dihydrocalysterol (9), petrosterol (7), and ficisterol (5).

Sterols of the cucurbitaceae

The sterol constituents of 32 seed and mature plant (leaves and stems, pericarp of the fruit and roots) materials from the 12 genera of the family Cucurbitaceae were investigated, and 44 sterols were identified or newly characterized. The sterols found were those possessing the following 7 types of skeletons: saturated, and Δ 5-, Δ 7-, Δ 8-, Δ 8(14)- and 14α-methyl-Δ 9(11)-unsaturated, and 14α-methyl-9β,19-cyclo skeletons, with the side chains of 24-unsubstituted, Δ 24-, 24-methyl-, 24-methyl-Δ 22-, 24-methylene-, 24-ethyl-, 24-ethyl-Δ 22-, 24-ethyl-Δ 24(250-, 24-ethylidene-, 24-ethyl-Δ 25- and 24-ethyl-δ 22,25-substituted, 24-methylene-25-methylated and 24,24-dimethylated (24,24-dimethyl-, 24,24-dimethyl-Δ 22- and 24,24-dimethyl-Δ 25-substituted) structures. The configurations at C-24 of the 24-alkylsterols were examined by high resolution 1H NMR and 13C NMR spectroscopy. The 24-methyl- and 24-ethylsterols lacking a Δ 25-bond examined (i.e. 24-methyl-, 24-methyl-Δ 22-, 24-ethyl- and 24-ethyl-Δ 22-sterols) were shown to occur as the C-24 epimeric mixtures, or as a single diastereoisomer, either 24α- and 24β-epimer. The 24-ethylsterols bearing a Δ 25-bond (i.e. 24-ethyl-δ 25- and 24-ethyl-Δ 22,25-sterols) were, on the other hand, composed of only 24β-epimers. The possible biosynthetic sequences to the cucurbitaceous sterols are discussed.

Differential effects of gabaculin and laevulinic acid on protochlorophyllide regeneration

Abstract The effects of gabaculin (3‐amino 2,3‐dihydrobenzoic acid) and laevulinic acid on the regeneration of protochlorophyllide from exogenous δ‐aminolaevulinic acid in leaves of dark‐grown barley (Hordeum vulgare) after a brief light treatment were compared. Gabaculin, a potent inhibitor of chlorophyll biosynthesis, did not inhibit this process showing that it affects the formation of δ‐aminolaevulinic acid rather than its further metabolism. Laevulinic acid, which is an inhibitor of δ‐aminolaevulinic acid dehydratase, prevented regeneration of protochlorophyllide provided pools of intermediates in the biosynthetic sequence were depleted. Formation of relatively large amounts of protochlorophyllide in some experiments suggests a lack of control in the utilization of δ‐aminolaevulinic acid for protochlorophyllide synthesis.

Enhanced production of dolichol, but not dolichyl phosphate, in the earliest stages of rat liver regeneration.

The regenerating liver presents a changed ability to use mevalonate 16 hr after partial hepatectomy. The dolichol content and its synthesis from mevalonate is increased, while no variation of dolichyl-P and ubiquinone parameters are detectable. The greater amount of mevalonate utilized to form dolichol, but not dolichyl-P, in this proliferating system, raises some questions about the physiological significance of these isoprenoid compounds and about their biosynthetic sequence.

Major Indole Alkaloids Produced in Cell Suspension Cultures of Rhazya strict a Decaisne

Eleven main alkaloids were identified from cell suspension cultures of Rhazya stricta grown in 4X-medium for 15 days. The alkaloids comprised the five groups Corynanthe, Strychnos, Eburnan, Secodine, and Aspidosperma and can be regarded as being typical Rhazya alkaloids, although the Strychnos alkaloid akuammicine has been isolated for the first time from the genus Rhazya. The most abundant compound was ( + )-1,2-dehydroaspidospermidine (15 mg/l medium) whereas all other constituents were synthesized in about 5 - 10 times lower amounts. More than 15 further alkaloids were formed as minor components which have not yet been identified. Cultivated Rhazya cells have been shown to be one of the richest alkaloid sources from apocynaceous cell suspensions. The isolated compounds, because of their structural heterogeneity, cannot be presently arranged into a scheme with coherent biosynthetic sequences.

Sequential appearance of radiolabel-tagged cyclohexadienyl derivatives of shikimate in Neurospora

The growing interest in the arogenate molecule and its derivatives in applied plant biochemistry stems from its regulation of the first enzyme in the aromatic pathway. The recent finding of a new derivative of arogenate (spiro-arogenate) in a mutant of Neurospora crassa raised the question of the biosynthetic sequence of cyclohexadienyl shikimate derived metabolites. Specifically labeled tritiated shikimate was used as a tracer. We have observed the uptake and metabolism of shikimate by this mutant. We have also demonstrated conclusively in vivo that arogenate appears prior to spiro-arogenate in this strain. The levels of accumulation of the various nonaromatic shikimate metabolites were determined in the cells. Intracellular arogenate levels exceeded prephenate levels which is exactly the opposite of results obtained in the culture supernate.