Mobilization and utilization of cyanogenic glycosides: the linustatin pathway

In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside linamarin (2-β-d-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the endosperm. After onset of germination, the cyanogenic diglucoside linustatin (2-[6-β-d-glucosyl-β-d-glucopyranosyloxy]-2- methylpropionitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The linustatin-splitting diglucosidase and the β-cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at this time. Based on these observations the following pathway for the in vivo mobilization and metabolism of cyanogenic glucosides is proposed: storage of monoglucosides (in the endosperm)—glucosylation—transport of the diglucoside (out of the endosperm into the seedling)—cleavage by diglucosidase—reassimilation of HCN to noncyanogenic compounds. The presence of this pathway demonstrates that cyanogenic glucosides, typical secondary plant products serve in the metabolism of developing plants as N-storage compounds and do not exclusively exhibit protective functions due to their repellent effect.     

Absence of cyanogenic glycosides in the tribe Miconieae (Melastomataceae)

In order to compare ant and non-ant defended species of Melastomataceae, production of hydrogen cyanide gas was tested in the field for 51 species of 10 genera of the tribe Miconieae. Using both the picric acid and the Feigl–Anger tests all populations surveyed tested negative for the presence of cyanogenic glycosides. These results confirm that cyanogenesis is rare in the family, although not completely absent. Cyanogenic glycosides are not responsible for the protection against herbivory in non-ant defended species, but this does not rule out that there are quantitative of qualitative differences in other secondary metabolites.     

Biosynthesis and regulation of cyanogenic glycoside production in forage plants

Applied Microbiology and Biotechnology - The natural products cyanogenic glycosides (CNglcs) are present in various forage plant species including Sorghum spp., Trifolium spp., and Lotus spp. The...     

Passisuberosin and epipassisuberosin: Two cyclopentenoid cyanogenic glycosides from Passiflora suberosa

A novel cyclopentenoid cyanogenic glycoside, passisuberosin (1-(β-D-glucopyranosyloxy)-4-hydroxy-2,3-epoxycyclopentanenitrile), has been isolated from Passiflora suberosa. Its structure was determined by means of ¹H NMR and ¹³C NMR and the identity of the glycosidic moieties by HPLC and TLC. A probable C-1 epimer, epipassisuberosin, was also present, as were smaller amounts of passicoriacin and epipassicoriacin, previously isolated from Passiflora coriacea. In addition, the presence of diglucosides ofpassisuberosin and epipassisuberosin was detected. These compounds differ in structure from those produced by other members of section Cieca, subgenus Plectostemma of Passiflora, the data suggest that the taxonomic placement of these two species should be re-evaluated.     

Biosynthesis of cyanogenic glucosides: in vitro analysis of the glucosylation step

The last step in the biosynthesis of cyanogenic glucosides, the glucosylation of the cyanohydrin intermediate, has been investigated in detail using Triglochin maritima seedlings. The glucosyltransferase activity is not associated with membranes and appears to be a "soluble" enzyme. The cyanohydrin intermediate, which is formed by hydroxylation of 4-hydroxyphenylacetonitrile by a membrane-bound enzyme, is free to equilibrate in the presence of the glucosyltransferase and UDPG, because it can be trapped very efficiently. This indicates that this intermediate is not channeled (unlike some of the other intermediates), although it is probably the most labile of all of them. The glucosyltransferase of T. maritima responsible for the glucosylation of the cyanohydrin was separated from another glucosyltransferase, which used 4-hydroxybenzylalcohol as a substrate, and purified over 200-fold. It catalyzed the glucose transfer from UDPG to only 4-hydroxymandelonitrile and 3,4-dihydroxymandelonitrile, giving rise to the respective cyanogenic glucosides. Although the activities with these two substrates behaved differently in certain respects (e.g., extent of inactivation during purification and difference in activation by higher salt concentrations), most of the data acquired favor the view that only one enzyme in T. maritima is responsible for the glucosylation of both substrates.     

Passibiflorin,epipassibiflorin and passitrifasciatin: cyclopentenoid cyanogenic glycosides from Passiflora

An epimeric mixture of two novel cyclopentenoid cyanogenic glycosides, passibiflorin [1-(6-O-β-D-rhamnopyranosyl-β-D-glucopyranosyloxy)-4-hydroxycyclopent-2-en-1-nitrile] and its C-1 epimer, epipassibiflorin, has been isolated from Passiflora biflora and P. talamancensis. The structures were determined by means of ¹H NMR and ¹³C NMR. Another novel cyclopentenoid cyanogenic glycoside, passitrifasciatin [1-(4-O-β-D-rhamnopyranosyl-β-D-glucopyranosyloxy)-4-hydroxycyclopent-2-en-1-nitrile] is described from Passiflora trifasciata. The structure was determined by means of ¹H NMR. The identification of the sugar moieties was made by HPLC and TLC. The isolation of a β-1 → 4 and a β-1 → 6-rhamnoglucoside of cyclopentenoid cyanogens from three species of subgenus Plectostemma of Passiflora suggests that diglycosides of this type are taxonomically diagnostic for the section.     

Gynocardin from Baileyoxylon lanceolatum and a revision of cyanogenic glycosides in Achariaceae

The cyclopentenone cyanhydrin glycoside gynocardin was the only cyanogen isolated from foliage of monotypic Australian rainforest tree, Baileyoxylon lanceolatum (Achariaceae). The presence of cyanogenic compounds in plants can have considerable taxonomic utility. A review of previous reports of cyanogenesis in the recently revised Achariaceae revealed distinct taxonomic patterns as well as inconsistencies in the reporting of cyanogenic compounds. This variation appears to be due to tissue level localisation of cyanogenic compounds as well as discrepancies in results obtained from different detection methods. Recommendations are made for future investigations.     

Positive effects of cyanogenic glycosides in food plants on larval development of the common blue butterfly

Cyanogenesis is a widespread chemical defence mechanism in plants against herbivory. However, some specialised herbivores overcome this protection by different behavioural or metabolic mechanisms. In the present study, we investigated the effect of presence or absence of cyanogenic glycosides in birdsfoot trefoil (Lotus corniculatus, Fabaceae) on oviposition behaviour, larval preference, larval development, adult weight and nectar preference of the common blue butterfly (Polyommatus icarus, Lycaenidae). For oviposition behaviour there was a female-specific reaction to cyanogenic glycoside content; i.e. some females preferred to oviposit on cyanogenic over acyanogenic plants, while other females behaved in the opposite way. Freshly hatched larvae did not discriminate between the two plant morphs. Since the two plant morphs differed not only in their content of cyanogenic glycoside, but also in N and water content, we expected these differences to affect larval growth. Contrary to our expectations, larvae feeding on cyanogenic plants showed a faster development and stronger weight gain than larvae feeding on acyanogenic plants. Furthermore, female genotype affected development time, larval and pupal weight of the common blue butterfly. However, most effects detected in the larval phase disappeared for adult weight, indicating compensatory feeding of larvae. Adult butterflies reared on the two cyanogenic glycoside plant morphs did not differ in their nectar preference. But a gender-specific effect was found, where females preferred amino acid-rich nectar while males did not discriminate between the two nectar mimics. The presented results indicate that larvae of the common blue butterfly can metabolise the surplus of N in cyanogenic plants for growth. Additionally, the female-specific behaviour to oviposit preferably on cyanogenic or acyanogenic plant morphs and the female-genotype-specific responses in life history traits indicate the genetic flexibility of this butterfly species and its potential for local adaptation.     

Biosynthesis of cyanogenic glucosides in Triglochin maritima and the involvement of cytochrome P450 enzymes.

The biosynthesis of the two cyanogenic glucosides, taxiphyllin and triglochinin, in Triglochin maritima (seaside arrow grass) has been studied using undialyzed microsomal preparations from flowers and fruits. Tyrosine was converted to p-hydroxymandelonitrile with V(max) and K(m) values of 36 nmol mg(-1) g(-1) fresh weight and 0.14 mM, respectively. p-Hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile accumulated as intermediates in the reaction mixtures. Using radiolabeled tyrosine as substrate, the radiolabel was easily trapped in p-hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile when these were added as unlabeled compounds. p-Hydroxyphenylacetaldoxime was the only product obtained using microsomes prepared from green leaves or dialyzed microsomes prepared from flowers and fruits. These data contrast earlier reports (H?sel and Nahrstedt, Arch. Biochem. Biophys. 203, 753-757, 1980; and Cutler et al., J. Biol. Chem. 256, 4253-4258, 1981) where p-hydroxyphenylacetaldoxime was found not to accumulate. All steps in the conversion of tyrosine to p-hydroxymandelonitrile were found to be catalyzed by cytochrome P450 enzymes as documented by photoreversible carbon monoxide inhibition, inhibition by antibodies toward NADPH-cytochrome P450 oxidoreductase, and by cytochrome P450 inhibitors. We hypothesize that cyanogenic glucoside synthesis in T. maritima is catalyzed by multifunctional cytochrome P450 enzymes similar to CYP79A1 and CYP71E1 in Sorghum bicolor except that the homolog to CYP71E1 in T. maritima exhibits a less tight binding of p-hydroxyphenylacetonitrile, thus permitting the release of this intermediate and its conversion into triglochinin.     

Biosynthesis of coumarin glycosides by transgenic hairy roots of Polygonum multiflorum

To investigate the substrate specificity and regio-selectivity of coumarin glycosyltransferases in transgenic hairy roots of Polygonum multiflorum, esculetin (1) and eight hydroxycoumarins (2-9) were employed as substrates. Nine corresponding glycosides (10-18) involving four new compounds, 6-chloro-4-methylcoumarin 7-O-β-D-glucopyranoside (15), 6-chloro-4-phenylcoumarin 7-O-β-D-glucopyranoside (16), 8-hydroxy-4-methylcoumarin 7-O-β-D-glucopyranoside (17), and 8-allyl-4-methylcoumarin 7-O-β-D-glucopyranoside (18), were biosynthesized by the hairy roots.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus

Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.     

Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300

Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through ¹H and ¹³C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated.     

Physiologically based pharmacokinetic modeling of hydrogen cyanide in humans following the oral administration of potassium cyanide and cyanogenic glycosides from food

Abstract

Cyanogenic glycosides (CGs) are commonly found in some edible plants and seeds. After ingestion, CGs can release toxic hydrogen cyanide (HCN) in humans. At present, unfortunately, there is no tool capable of predicting the cyanide concentration in human blood and organs following oral administration of CG-containing food. The aim of this study was to develop a physiologically based pharmacokinetic (PBPK) model of cyanide following the ingestion of CG-containing food in humans. To develop this model, pharmacokinetic data concerning cyanide concentration levels in humans exposed to potassium cyanide (KCN) and CG-containing foods (persipan paste, linseed, cassava, and bitter apricot kernels) were obtained from published data. This study created a model structure consisting of four organ compartments including the lungs, kidneys, liver, and slowly perfused tissues by employing Berkeley Madonna software to extract three unknown parameters including the maximum velocity of rhodanese, the absorption rate constant, and the bioavailability for oral administration of KCN and the four CG-containing foods (equivalent a 6.8?mg dose of cyanide). The model was then validated by comparing the simulated results for the concentration-time courses of cyanide levels in venous blood with data from two clinical studies covering the oral administration of KCN and linseed at three other doses.     

Biosynthesis of oleanolic acid glycosides by subcellular fractions of Calendula officinalis seedlings

The synthesis of oleanolic acid 3β-d-glucuronoside from oleanolic acid and UDPGlcA has been demonstrated in cell-free preparations from C. officinalis seedlings. Moreover, the formation of more complex glycosides by successive additions of galactose and glucose to oleanolic acid glucuronoside was observed when cell-free preparations were incubated with UDPGal or UDPGlc. The consecutive steps of oleanolic acid glycosylation are localized in three different cellular compartments. The biosynthesis of the 3-glucuronoside takes place in the microsomes, the elongation of the sugar chain at C-3 of the aglycone proceeds in heavy membrane structures which are probably fragments of the Golgi complex while a cytosol enzyme(s) is involved in glucosylation of the C-17 carboxyl group of oleanolic acid.     

Biosynthesis of oleanolic acid glycosides in protoplasts isolated from Calendula officinalis L. roots

Many medicinal plants contain oleanane saponins in roots, however, only scarce data on their biosynthesis in this organ are available so far, including our previous results concerning Calendula officinalis plant. Thus, the purpose of the present work was to confirm the presumable biosynthetic pathway of oleanolic acid glycosides in roots of young C. officinalis plants. First of all, the effective method of isolation of protoplasts from C. officinalis roots was established. Then, isolated root protoplasts were supplied with radioactive precursors, [2-¹⁴C] mevalonate (MVA) and [3-³H] oleanolic acid (OL) and their transformations were studied with comparison to results obtained with excised roots. The penetration of both precursors into protoplasts was more rapid and effective than in the case of excised roots. The labeling of sterols and OL during the incubation with MVA showed that the isoprenoid pathway leading to triterpenoids was operative in excised roots as well as isolated root protoplasts. Moreover, the transformations of OL into two series of its glycosides, i.e. glucosides and glucuronides were investigated. It has been shown that both series of OL glycosides are synthesized in isolated root protoplasts in the same way as in excised roots of young marigold plants.