Phytochemistry (v.65, #3)

Graphical Abstracts (245-247).

Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.Horseradish peroxidase is one of the most important enzymes obtained from a plant source. It continues to attract the attention of researchers from a variety of disciplines because of its practical and commercial applications. Advances in understanding the structure and catalytic mechanism of horseradish peroxidase have been made using protein engineering and other techniques. The physiological role of the enzyme is now being investigated in the context of new information on the plant peroxidase gene family of Arabidopsis thaliana.
Keywords: Horseradish peroxidase; Armoracia rusticana; Arabidopsis thaliana; Cruciferae; Heme; Hydrogen peroxide; Indole-3-acetic acid; Protein engineering;

On the origins of triterpenoid skeletal diversity by Ran Xu; Gia C. Fazio; Seiichi P.T. Matsuda (261-291).
The triterpenoids are a large group of natural products derived from C30 precursors. Nearly 200 different triterpene skeletons are known from natural sources or enzymatic reactions that are structurally consistent with being cyclization products of squalene, oxidosqualene, or bis-oxidosqualene. This review categorizes each of these structures and provides mechanisms for their formation.The triterpenoids are a large group of natural products derived from C30 precursors. Nearly 200 different triterpene skeletons are known from natural sources or enzymatic reactions that are structurally consistent with being cyclization products of squalene, oxidosqualene, or bis-oxidosqualene. This review categorizes each of these structures and provides mechanisms for their formation.
Keywords: Triterpene; Triterpenoid; Oxidosqualene cyclization; Squalene; Cyclization; Isoprene rule;

Cyanogenic glucosides and plant–insect interactions by Mika Zagrobelny; Søren Bak; Anne Vinther Rasmussen; Bodil Jørgensen; Clas M. Naumann; Birger Lindberg Møller (293-306).
Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are considered to have an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their predator defense. A few species of Arthropoda (within Diplopoda, Chilopoda, Insecta) are able to de novo synthesize cyanogenic glucosides and, in addition, some of these species are able to sequester cyanogenic glucosides from their host plant (Zygaenidae). Evolutionary aspects of these unique plant–insect interactions with focus on the enzyme systems involved in synthesis and degradation of cyanogenic glucosides are discussed.Cyanogenic glucosides are phytoanticipins important in plant defense against herbivory. Some specialized herbivores are able to degrade, de novo synthesize, and/or sequester cyanogenic glucosides from host plants. Co-evolutionary aspects of these complex interactions are discussed.
Keywords: Cyanogenic glucosides; Cyanogenesis; Linamarin; Lotaustralin; Lepidoptera; Zygaenidae; Papilionoidea;

Purification and identification of a Ca2+-pectate binding peroxidase from Arabidopsis leaves by Kavita Shah; Claude Penel; Jean Gagnon; Christophe Dunand (307-312).
A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca2+-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca2+-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca2+-pectate structure is discussed.AtPrx34 peroxidase obtained from Arabidopsis leaf extract was purified by affinity chromatography through a Ca2+-pectate/polyacrylamide gel and microsequenced. Atprx34 transcripts accumulated in root, stem, flower and leaf. Recombinant Atprx34 exhibited an affinity for Ca2+-pectate, possibly through an amino acid motif consisting in K233, R225 and K242.
Keywords: Peroxidase; Arabidopsis Ca2+-pectate complex;

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins by Catherine Lapierre; Gilles Pilate; Brigitte Pollet; Isabelle Mila; Jean-Charles Leplé; Lise Jouanin; Hoon Kim; John Ralph (313-321).
Thioacidolysis produces marker compounds revealing the incorporation of sinapaldehyde into lignins of CAD-deficient poplars, the marker level reflecting the degree of CAD-suppression. The most severely depressed lines had ca. 50% of their lignins soluble in alkali at 37 °C.A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8–O–4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and β–O–4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.
Keywords: Poplar (Populus deltoides×Populus nigra; cv Ogy); Salicaceae; Lignin structure; Lignin biosynthesis; Cinnamyl alcohol dehydrogenase; Transgenic; Thioacidolysis; Sinapaldehyde; Coniferaldehyde;

Compound-specific carbon and hydrogen isotopic compositions of lipid biomolecules (n-alkanes, n-alkanoic acids, n-alkanols, sesquiterpenes, diterpenes, phytol, diterpenols and β-sitosterol), extracted from Cryptomeria japonica leaves, were determined in order to understand isotopic fractionations occurring during lipid biosynthesis in this species. All lipid biomolecules were depleted in both 13C and D relative to bulk tissue and ambient water, respectively. n-Alkyl lipids associated with the acetogenic pathway were depleted in 13C relative to bulk tissue by 2.4–9.9‰ and depleted in D relative to ambient water by 91–152‰. C15- and C30-isoprenoid lipids (sesquiterpenes, squalene and β-sitosterol) associated with the mevalonic-acid pathway are depleted in 13C relative to bulk tissue by 1.7–3.1‰ and depleted in D relative to ambient water by 212–238‰. C20-isoprenoid lipids (phytol and diterpenoids) associated with the non-mevalonic-acid pathway were depleted in 13C relative to bulk tissue by 4.6–5.9‰ and depleted in D relative to ambient water by 238–303‰. Phytol was significantly depleted in D by amounts up to 65‰ relative to other C20 isoprenoid lipids. The acetogenic, mevalonic-acid and non-mevalonic-acid pathways were clearly discriminated using a cross-plot between the carbon and hydrogen isotopic fractionations.Compound-specific carbon and hydrogen isotopic compositions of lipid biomolecules (n-alkanes, n-alkanoic acids, n-alkanols, sesquiterpenes, diterpenes, phytol, diterpenols and β-sitosterol) have been dtermined in Cryptomeria japonica (C3-gymnosperm cedar) leaves.
Keywords: Cryptomeria japonica; Taxodiaceac; δD; δ 13C; Isotopic fractionation; Lipid biosynthesis; Acetogenic; Mevalonic-acid; Methylerythritol-phosphate;

The anti-staphylococcal activity of Angelica dahurica (Bai Zhi) by Doris Lechner; Michael Stavri; Moyosoluwa Oluwatuyi; Rogelio Pereda-Miranda; Simon Gibbons (331-335).
Bioassay-guided isolation of a hexane extract of the roots of Angelica dahurica (Apiaceae) led to the isolation of 3(R), 8(S)-falcarindiol as the active anti-bacterial principle. This compound displayed minimum inhibitory concentrations of 8–32 μg/ml against multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA).Bioassay-guided fractionation of a hexane extract prepared from the roots of the Chinese drug Angelica dahurica (Bai Zhi) led to the isolation of the polyacetylenic natural product falcarindiol (1). The absolute stereochemistry of this compound was confirmed by careful 1H NMR analysis of its (R)- and (S)-Mosher ester derivatives as the 3(R), 8(S) isomer. Activity was tracked using a Mycobacterium fortuitum screening assay and the purified product was evaluated against multidrug-resistant and methicillin-resistant strains of Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) of this metabolite ranged from 8 to 32 μg/ml highlighting the potential of the acetylene natural product class as antibiotic-lead compounds. These MIC values compare favourably with some of the newest agents in development for the treatment of MRSA infection and indicate that further evaluation of the antibiotic activity of acetylenes is warranted.
Keywords: Bai Zhi; Angelica dahurica; Apiaceae; Mosher's esters; Multidrug-resistance; Staphylococcus aureus; MDR; MRSA; Polyacetylenes;

Generation of ginsenosides Rg3 and Rh2 from North American ginseng by David G Popovich; David D Kitts (337-344).
Rg3 and Rh2 ginsenosides are primarily found in Korean red ginseng root (Panax ginseng C.A. Meyer) and valued for their bioactive properties. We quantified both Rh2 and Rg3 ginseng leaf and Rg3 from root extracts derived from North American ginseng (Panax quinquefolius). Quantification was obtained by application of HPLC with ion fragments detected using ESI-MS. Ginseng leaf contained 11.3±0.5 mg/g Rh2 and 7.5±0.9 mg/g Rg3 in concentrated extracts compared to 10.6±0.4 mg/g Rg3 in ginseng root. No detectable Rh2 was found in root extracts by HPLC, although it was detectable by ESI-MS analysis. Ginsenosides Rg3 and Rh2 were detected following hot water reflux extraction, but not from tissues extracted with 80% aqueous ethanol at room temperature. Therefore ginsenosides Rg3 and Rh2 are not naturally present in North American ginseng, but are products of a thermal process. Using ESI-MS analysis, it was found that formation of Rg3 and Rh2, among other compounds, were a function of heating time and were breakdown products of the more abundant ginsenosides Rb1 and Rc. Our findings that heat processed North American ginseng leaf is an excellent source of Rh2 ginsenoside is an important discovery considering that ginseng leaf material is obtainable throughout the entire plant cycle for recovery of valuable ginsenosides for pharmaceutical use.Ginsenoside Rh2 is formed as a result of thermal extraction of North American ginseng (Panax quinquefolius).
Keywords: Panax quinquefolius; Araliaceae; North American ginseng; ginseng leaf; ginseng root; ginsenosides; Rh2; Rg3; LC/MS;

Potential cancer chemopreventive constituents of the leaves of Macaranga triloba by Dae Sik Jang; Muriel Cuendet; Alison D Pawlus; Leonardus B.S Kardono; Kazuko Kawanishi; Norman R Farnsworth; Harry H.S Fong; John M Pezzuto; A.Douglas Kinghorn (345-350).
Activity-guided fractionation of the leaves of Macaranga triloba, using an in vitro bioassay based on the inhibition of cyclooxygenase-2, resulted in the isolation of a rotenoid, 4,5-dihydro-5′α-hydroxy-4′α-methoxy-6a,12a-dehydro-α-toxicarol (1), as well as 12 known compounds, (+)-clovan-2β,9α-diol, ferulic acid, 3,7,3′,4′-tetramethylquercetin, 3,7,3′-trimethylquercetin, 3,7-dimethylquercetin, abscisic acid, 1β,6α-dihydroxy-4(15)-eudesmene, 3β-hydroxy-24-ethylcholest-5-en-7-one, loliolide, scopoletin, taraxerol, and 3-epi-taraxerol. The structure of compound 1 was determined using spectroscopic methods. All isolates were evaluated for their potential to inhibit cyclooxygenases-1 and -2 by measuring PGE2 production, and to induce quinone reductase in cultured Hepa 1c1c7 mouse hepatoma cells.A new rotenoid, 4,5-dihydro-5′α-hydroxy-4′α-methoxy-6a,12a-dehydro-α-toxicarol (1), together with twelve known compounds, was isolated from the leaves of Macaranga triloba by in vitro bioassay-guided fractionation based on the inhibition of cyclooxygenase-2.
Keywords: Macaranga triloba; Euphorbiaceae; Flavonoids; 4,5-Dihydro-5′α-hydroxy-4′α-methoxy-6a,12a-dehydro-α-toxicarol; Cyclooxygenases-1 and -2; Quinone reductase;

The rapidly growing, nearly achlorophyllous, young leaves of Inga umbellifera express high concentrations of mono and dimeric 3-O-gluco-cinnamoyl catechin/epicatechin, rare forms of substituted flavan-3-ols. Here we present structures for five novel compounds in this class: three monomers [catechin-3-O-β-d-gluco(2-cinnamoyl)pyranoside, catechin-3-O-β-d-gluco(6-cinnamoyl) pyranoside, catechin-3-O-β-d-gluco(2,6-biscinnamoyl)pyranoside] and two dimeric procyanidins [catechin-3-O-β-d-glucopyrano-(4α→8)-catechin-3-O-β-d-gluco(2-cinnamoyl)pyranoside and catechin-3-O-β-d-glucopyrano-(4α→8)-epicatechin-3-O-β-d-gluco(6-cinnamoyl)pyranoside]. The young leaves of Inga umbellifera express high concentrations of 3-O-(cinnamoyl)glucosides of catechin and epicatechin.The young leaves of Inga umbellifera express high concentrations of 3-O-(cinnamoyl)glucosides of catechin and epicatechin.
Keywords: Inga umbellifera; Fabaceae; Young leaf secondary chemistry; Acetylated flavan-3-ol glycoside; Acetylated procyanidin glycoside; Cinnamoyl glycoside;

Oligomeric hydrolyzable tannins from Monochaetum multiflorum by José H Isaza; Hideyuki Ito; Takashi Yoshida (359-367).
Four hydrolyzable tannins, nobotanins Q, R, S, and T, were isolated from the aqueous acetone extract of the dried leaves of Monochaetum multiflorum (Melastomataceae), a plant indigenous to Colombia. Their dimeric and tetrameric structures were elucidated by spectral and chemical methods. Eight known hydrolyzable tannin monomers and eight ellagitannin oligomers characteristic of melastomataceous plants were also characterized as tannin constituents of the plant.From leaves of Monochaetum multiflorum (Melastomataceae), four ellagitannin oligomers, nobotanins Q, R, S and, T were isolated and their structures were determined by detailed NMR analyses.
Keywords: Monochaetum multiflorum; Melastomataceae; Tannins; Oligomeric ellagitannin; Nobotanins;

Erratum to “Cell wall-associated enzymes in fungi” [Phytochemistry 64 (2003) 339–366] by Dora M. Rast; Daniel Baumgartner; Christoph Mayer; G.O. Hallenstein (369-370).