Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with ¹³C-labelled precursors in an ¹⁸O₂ atmosphere to observe incorporation of the two isotopes at defined biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composition of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosynthetic stages in the sequence O-1 → O-2 → O-5. In addition, the incubation of W. thyrsiflora root cultures with ¹³C-labelled lachnanthocarpone established a direct biosynthetic precursor–product relationship with 1,2,5,6-tetraoxygenated phenylphenalenones.     

Oxygen fixation into hydroxyproline in etiolated maize seedlings : verification by tandem mass spectrometry

Etiolated maize (Zea mays L.) seedlings were grown in the dark for 5 days in an atmosphere enriched with 10.0 atom% ¹⁸O₂. Hydroxyproline was isolated from root and shoot tissues, purified, and methylated. It was not possible to determine ¹⁸O incorporation into hydroxyproline by conventional mass spectrometry because the final product was not sufficiently pure. The final product was analyzed successfully by tandem mass spectrometry. The ¹⁸O content of the hydroxyl oxygen atom was 10 ± 0.7 atom%. This result demonstrates that the hydroxyl oxygen atom in hydroxyproline was derived exclusively from molecular oxygen.     

Isolation and structure of d-xylans from pericarp seeds of Opuntia ficus-indica prickly pear fruits

Xylans were isolated from the pericarp of prickly pear seeds of Opuntia ficus-indica (OFI) by alkaline extraction, fractionated by precipitation and purified. Six fractions were obtained and characterized by sugar analysis and NMR spectroscopy. They were assumed to be (4-O-methyl-d-glucurono)-d-xylans, with 4-O-α-d-glucopyranosyluronic acid groups linked at C-2 of a (1→4)-β-d-xylan. The sugar composition and the ¹H and ¹³C NMR spectra showed that their chemical structures were very similar, but with different proportions of d-Xyl and 4-O-Me-d-GlcA. Our results showed that, on average, the water soluble xylans have one nonreducing terminal residue of 4-O-methyl-d-glucuronic acid for every 11 to 14 xylose units, whereas in the water non-soluble xylans, xylose units can varied from 18 to 65 residues for one nonreducing terminal residue of 4-O-methyl-d-glucuronic acid.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

Structures of the O-antigens of Escherichia coli O13, O129, and O135 related to the O-antigens of Shigella flexneri

O-Polysaccharides (O-antigens) were isolated from Escherichia coli O13, O129, and O135 and studied by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy. They were found to possess a common →2)-l-Rha-(α1→2)-l-Rha-(α1→3)-l-Rha-(α1→3)-d-GlcNAc-(β1→ backbone, which is a characteristic structural motif of the O-polysaccharides of Shigella flexneri types 1-5. In both the bacterial species, the backbone is decorated with lateral glucose residues or/and O-acetyl groups. In E. coli O13, a new site of glycosylation on 3-substituted Rha was revealed and the following O-polysaccharide structure was established:The structure of the E. coli O129 antigen was found to be identical to the O-antigen structure of S. flexneri type 5a specified in this work and that of E. coli O135 to S. flexneri type 4b reported earlier.     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

O-Methylation of phenylphenalenone phytoalexins in Musa acuminata and Wachendorfia thyrsiflora

Biosynthetic O-methylation at various sites along the backbone of inducible phenylphenalenones in Musa acuminata var. “Williams” (Musaceae) and Wachendorfia thyrsiflora (Haemodoraceae) was investigated using ¹³C-labelled precursors. The inducibility of O-methylated metabolites was demonstrated in both species and the origin of methoxyl group from [methyl-¹³C]l-methionine was confirmed. In addition to known phenylphenalenones, a methoxylated metabolite, 4-(4-hydroxy-3-methoxy-phenyl)-benzo[de]isochromene-1,3-dione, was detected and its structure elucidated mainly by NMR spectroscopic techniques. The experiments were used to discriminate methionine-derived and artificial methoxy groups formed during methanolic extraction. Finally, demethylation of 4′-methoxycinnamic acid and subsequent conversion to 3′,4′-methylenedioxycinnamic acid was demonstrated in M. acuminata.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

Hydroperoxide bicyclase CYP50918A1 of Plasmodiophora brassicae (Rhizaria,SAR): Detection of novel enzyme of oxylipin biosynthesis

The genome of the cabbage clubroot pathogen Plasmodiophora brassicae Woronin 1877 (Cercozoa, Rhizaria, SAR), possesses two expressed genes encoding the P450s that are phylogenetically related to the enzymes of oxylipin biosynthesis of the CYP74 clan. The cDNA of one of these genes (CYP50918A1) has been expressed in E. coli. The preferred substrate for the recombinant protein, the 13-hydroperoxide of α-linolenic acid (13-HPOT), was converted to the novel heterobicyclic oxylipins, plasmodiophorols A and B (1 and 2) at the ratio ca. 12:1. Compounds 1 and 2 were identified as the substituted 6-oxabicyclo[3.1.0]hexane and 2-oxabicyclo[2.2.1]heptane (respectively) using the MS and NMR spectroscopy, as well as the chemical treatments. The ¹⁸O labelling experiments revealed the incorporation of a single ¹⁸O atom from [¹⁸O₂]13-HPOT into the epoxide and ether functions of products 1 and 2 (respectively), but not into their OH groups. In contrast, the ¹⁸O from [¹⁸O₂]water was incorporated only into the hydroxyl functions. One more minor polar product, plasmodiophorol C (3), identified as the cyclopentanediol, was formed through the hydrolysis of compounds 1 and 2. Plasmodiophorols A–C are the congeners of egregiachlorides, hybridalactone, ecklonialactones and related bicyclic oxylipins detected before in some brown and red algae. The mechanism of 13-HPOT conversions to plasmodiophorols A and B involving the epoxyallylic cation intermediate is proposed. The hydroperoxide bicyclase CYP50918A1 is the first enzyme controlling this kind of fatty acid hydroperoxide conversion.     

Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain

Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (−)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used ¹⁸O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under ¹⁸O₂, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H₂¹⁸O. Pisatin synthesized by pea pods in the presence of ¹⁸O₂ or H₂¹⁸O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H₂O and not from O₂. Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.     

Biosynthesis of the iridoid glucoside cornin in Verbena officinalis

The biosynthesis of cornin (verbenalin) and dihydrocornin in Verbena officinalis has been investigated. The incorporation of [²H] deoxyloganin was found to be largely independent of the incubation time between one day and a week. An improved method for the preparation of deoxygeniposide from gardenoside is reported and [²H]-iridodial glucoside and [²H]-iridotrial glucoside were prepared from the former. Feeding experiments with young plants using these glucosides, as well as the aglucones, showed much better incorporations for the latter compounds as measured by ²H NMR spectroscopy. ¹³ C-labelled 10-hydroxygeraniol, 10-hydroxycitronellol, iridodial, iridotrial, iridodial glucoside, iridotrial glucoside, deoxyloganic acid, and deoxyloganic acid aglucone were prepared. [¹³C]-Mevalonic acid and the above compounds were fed to plants of medium age, and all gave incorporations measurable by ¹³C NMR spectroscopy into dihydrocornin. The postulated existence of two different metabolic pathways in the biosynthesis of cornin in young and old plants, respectively, could not be established as complete scrambling between the C-3 and C-11 in the iridoid skeleton apparently takes place with all the early precursors. The complete pathway from iridodial forwards to hastatoside has been elucidated.     

Paleosalinity in a brackish lake during the Holocene based on stable oxygen and carbon isotopes of shell carbonate in Nakaumi Lagoon, southwest Japan

Based on the relationship between salinity and δ¹⁸O and δ¹³C of modern shells in the Lake Nakaumi-Shinji lagoon system (southwestern Japan), where the salinity changes regularly from ca. 1 PSU to 34 PSU, a paleosalinity record for Nakaumi Lagoon during the Holocene has been derived from bulk mollusk shell δ¹⁸O and δ¹³C data. The robust relationships between the salinity and modern shell δ¹⁸O_ar and δ¹³C_ar (aragonite) were used to calibrate the paleosalinity reconstruction. The salinity relationships are expressed by the regressions:

Salinity (PSU)=3.86 δ¹⁸O_ar(‰ VPDB)+33.9 (n=18, r=0.978)     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

A new naturally acetylated triterpene saponin from Nigella sativa

A new glycosylated triterpene 1 was identified as 3-O-[β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl]-11-methoxy-16-hydroxy-17-acetoxy hederagenin from an ethanolic extract of seeds of Nigella sativa Linn. Identification of the naturally acetylated saponin was based on chemical and spectroscopic analyses including FABMS, ¹H, ¹³C, and 2D NMR and DEPT. The saponin was a penta hydroxy pentacyclic triterpene, in which one hydroxyl group was acetylated and other one was methylated naturally.     

NMR studies of the conformation of the natural sweetener rebaudioside A

Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four β-d-glucopyranose units are attached to the aglycone steviol. Its ¹H and ¹³C NMR spectra in pyridine-d₅ were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the β-d-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.     

Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution

The structure of the disaccharide cellulose subunit cellobiose (4-O-β-D-glucopyranosyl-D-glucose) in solution has been determined via neutron diffraction with isotopic substitution (NDIS), computer modeling and nuclear magnetic resonance (NMR) spectroscopic studies. This study shows direct evidence for an intramolecular hydrogen bond between the reducing ring HO3 hydroxyl group and the non-reducing ring oxygen (O5′) that has been previously predicted by computation and NMR analysis. Moreover, this work shows that hydrogen bonding to the non-reducing ring O5′ oxygen is shared between water and the HO3 hydroxyl group with an average of 50% occupancy by each hydrogen-bond donor. The glycosidic torsion angles φ_H and ψ_H from the neutron diffraction-based model show a fairly tight distribution of angles around approximately 22^° and −40^°, respectively, in solution, consistent with the NMR measurements. Similarly, the hydroxymethyl torsional angles for both reducing and non-reducing rings are broadly consistent with the NMR measurements in this study, as well as with those from previous measurements for cellobiose in solution.     

Structure of the O-polysaccharides of the lipopolysaccharides of Mesorhizobium loti HAMBI 1148 and Mesorhizobium amorphae ATCC 19655 containing two O-methylated monosaccharides

The O-polysaccharide of Mesorhizobium loti HAMBI 1148 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation, and ¹H and ¹³C NMR spectroscopies, including 2D ¹H/¹H COSY, TOCSY, ROESY, and H-detected ¹H/¹³C HSQC experiments. The O-polysaccharide was found to have a branched hexasaccharide-repeating unit of the following structure:where 2-acetamido-2-deoxy-4-O-methyl-d-glucose (d-GlcNAc4Me) and methyl group on 2-substituted d-rhamnose (Me) shown in italics are present in ∼80% and ∼40% repeating units, respectively. Similar studies of the O-polysaccharide from Mesorhizobium amorphae ATCC 19655 by sugar analysis and NMR spectroscopy revealed essentially the same structure but a higher content of 3-O-methyl-d-rhamnose (∼70%).     

Phenolic constituents of Gnaphalium uliginosum L.

The herb Gnaphalium uliginosum L. is an annual plant widely used in Russian and Bulgarian phytotherapy in the treatment of hypertension, thrombophlebitis, phlebothrombosis and ulcers. Decoction and infusion of G. uliginosum are known to possess anti-inflammatory, astringent, and antiseptic properties. Oil extracts are used in the treatment of laryngitis, upper respiratory catarrh and tonsillitis. However, there is still lack of information about the active compounds.Ten phenolic compounds have been identified from the aerial parts of G. uliginosum including seven flavonoid glucosides and three phenylpropanoids. Their chemical structures were elucidated on the basis of 1D and 2D NMR and HRESIMS. Among the identified compounds the first full assignments of the ¹H and ¹³C NMR spectra of, 5,7,4′-trihydroxy-6,3′-dimethoxyflavone-7-O-β-d-(6″-O-caffeoyl)-glucopyranoside are firstly reported in this paper.     

Source of oxygen in cytochrome P-450 catalyzed carbinolamine formation

N-Methylcarbazole was incubated in H₂O¹⁸ and under an ¹⁸O atmosphere. N-Hydroxymethylcarbazole, 1-OH- and 3-OH-N-methylcarbazole were isolated by HPLC and analyzed for ¹⁸O content In incubations containing ¹⁸O, all three metabolites showed >95% ¹⁸O incorporation. In incubations containing H₂O¹⁸, the N-hydroxymethyl metabolite showed ¹⁶O incorporation equal to control incubations in 100% H₂O. These data demonstrate that the sole source of oxygen in cytochrome P-450 catalyzed, NADPH supported N-hydroxymethylcarbazole formation is atmospheric oxygen.