Phytochemistry (v.70, #3)

Plant glycosidases acting on protein-linked oligosaccharides by Renaud Léonard; Richard Strasser; Friedrich Altmann (318-324).
This review covers plant glycosidases acting on N-glycoproteins or arabinogalactan proteins whose genes have been identified and discusses physiological roles or in vitro uses.Glycosidases have been used as invaluable tools in glycobiology research for decades, and their role in glycoprotein maturation has been amply studied. The molecular biological coverage of this large group of enzymes has only recently reached an appreciable level. In this review, we present an overview of plant glycosidases, whose DNA/protein sequence has been identified and for which recombinant enzymes have been characterized. The physiological role in the maturation of glycoproteins is discussed as well as the biotechnological prospects arising from knowing the enzymes responsible for the removal of terminal N-acetylglucosamine residues. The current knowledge on plant fucosidases and of the first bits of information on glycosidases acting on arabinogalactan proteins is presented.
Keywords: Glycosidase; Arabinogalactan protein; N-glycan; Glycoprotein;

Plant UGTs glycosylate a vide range of acceptor molecules using several different sugars. This review summarizes current knowledge of the relationship between 3D structure and substrate specificity and examines the accuracy of plant UGT structures derived from homology modeling by comparison to the crystal structures currently available.Plant family 1 UDP-dependent glycosyltransferases (UGTs) catalyze the glycosylation of a plethora of bioactive natural products. In Arabidopsis thaliana, 120 UGT encoding genes have been identified. The crystal-based 3D structures of four plant UGTs have recently been published. Despite low sequence conservation, the UGTs show a highly conserved secondary and tertiary structure. The sugar acceptor and sugar donor substrates of UGTs are accommodated in the cleft formed between the N- and C-terminal domains. Several regions of the primary sequence contribute to the formation of the substrate binding pocket including structurally conserved domains as well as loop regions differing both with respect to their amino acid sequence and sequence length. In this review we provide a detailed analysis of the available plant UGT crystal structures to reveal structural features determining substrate specificity. The high 3D structural conservation of the plant UGTs render homology modeling an attractive tool for structure elucidation. The accuracy and utility of UGT structures obtained by homology modeling are discussed and quantitative assessments of model quality are performed by modeling of a plant UGT for which the 3D crystal structure is known. We conclude that homology modeling offers a high degree of accuracy. Shortcomings in homology modeling are also apparent with modeling of loop regions remaining as a particularly difficult task.
Keywords: UGT; Glycosyltransferase; Substrate specificity; Sugar donor specificity; Crystal structure; Homology modeling;

The expression of two ACO genes, in addition to the well characterized MD-ACO1, and their protein products is reported. While MD-ACO1 is expressed in mature fruit, the other ACC oxidase genes are also expressed in fruit and in leaf tissues and the significance for ethylene production in apple is discussed.Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K cat) of 6.6 × 10−2 (MD-ACO1), 3.44 × 10−2 (Md-ACO2) and 9.14 × 10−2 (MD-ACO3) and K cat/K m (μM s−1) values of 7.38 × 10−4  μM s−1 (MD-ACO1), 0.86 × 10−4  M s−1 (MD-ACO2) and 3.8 × 10−4  μM s−1 (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.
Keywords: Malus domestica; ACC oxidase; Apple fruit development; Apple leaf development; Ethylene;

In many plants, multiple forms of phospholipase D have been found. Studies on the specificity of two isoenzymes from white cabbage and two ones from opium poppy suggest a physiological relevance of the different hydrolytic and transphosphatidylation activities.Phospholipase D (PLD) catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond. In many plants, several isoforms of PLD have been identified without knowing their functional differences. In this paper, the specificities of two PLD isoenzymes from white cabbage (Brassica oleracea var. capitata) and two ones from opium poppy (Papaver somniferum L.), which were recombinantly produced in Escherichia coli, were compared in the hydrolysis of phospholipids with different head groups and in the transphosphatidylation of phosphatiylcholine with several acceptor alcohols. In a biphasic reaction system, consisting of buffer and diethyl ether, the highly homologous isoenzymes are able to hydrolyze phosphatidylcholine, -glycerol, -ethanolamine, -inositol and – with one exception – also phosphatidylserine but with different individual reaction rates. In transphosphatidylation of phosphatidylcholine, they show significant differences in the rates of head group exchange but with the same trend in the preference of acceptor alcohols (ethanolamine > glycerol ≫  l-serine). For l- and d-serine a stereoselectivity of PLD was observed. The results suggest a physiological relevance of the different hydrolytic and transphosphatidylation activities in plant PLD isoenzymes.
Keywords: Phospholipase D; Plant isoenzymes; Phospholipid hydrolysis; Transphosphatidylation; d-Serine; Enantiomer specificity; White cabbage; Opium poppy;

Functional analysis of a putative second terpene synthase (TPS) divalent metal binding motif is presented. Specifically, to probe the surprising observation that plant TPS occasionally contain Gly in place of an otherwise conserved Thr/Ser that has been observed to directly ligate a divalent metal ion.Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this ‘middle’ position of the putative second divalent metal binding motif in plant TPS. Herein we report mutational investigation of this discrepancy in a model plant diterpene cyclase, abietadiene synthase from Abies grandis (AgAS). Substitution of the corresponding Thr in AgAS with Ser or Gly decreased catalytic activity much less than substitution with Ala. We speculate that the ability of Gly to partially restore activity relative to Ala substitution for Ser/Thr stems from the associated reduction in steric volume enabling a water molecule to substitute for the hydroxyl group from Ser/Thr, potentially in a divalent metal ion coordination sphere. In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS.
Keywords: Abies grandis; Terpene synthase; Enzymatic mechanism; Cyclization; Metal binding motifs; Labdane-related diterpenoids;

Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid by Yoshiya Seto; Shigeki Hamada; Hideyuki Matsuura; Mana Matsushige; Chizuru Satou; Kosaku Takahashi; Chikara Masuta; Hiroyuki Ito; Hirokazu Matsui; Kensuke Nabeta (370-379).
The universal existence of 12-hydroxyjasmonic acid (tuberonic acid, TA) and its glucoside (TAG) and TA glucosyltranferase activity are demonstrated. The glucosyltranferase, which is active toward TA, was purified from rice cell cultures and characterized as a putative salicylic acid (SA) glucosyltransferase (OsSGT). OsSGT was active toward TA and also SA. Expression analysis of mRNA of OsSGT established that its gene expression was induced by wounding stress.Tuberonic acid (12-hydroxy epi-jasmonic acid, TA) and its glucoside (TAG) were isolated from potato leaflets (Solanum tuberosum L.) and shown to have tuber-inducing properties. The metabolism of jasmonic acid (JA) to TAG in plant leaflets, and translocation of the resulting TAG to the distal parts, was demonstrated in a previous study. It is thought that TAG generated from JA transmits a signal from the damaged parts to the undamaged parts by this mechanism. In this report, the metabolism of TA in higher plants was demonstrated using [12-3H]TA, and a glucosyltransferase active toward TA was purified from the rice cell cultures. The purified protein was shown to be a putative salicylic acid (SA) glucosyltransferase (OsSGT) by MALDI-TOF-MS analysis. Recombinant OsSGT obtained by overexpression in Escherichia coli was active not only toward TA but also toward SA. The OsSGT characterized in this research was not specific, but this is the first report of a glucosyltransferase active toward TA. mRNA expressional analysis of OsSGT and quantification of TA, TAG, SA and SAG after mechanical wounding indicated that OsSGT is involved in the wounding response. These results demonstrated a crucial role for TAG not only in potato tuber formation, but also in the stress response in plants and that the SA glucosyltransferase can work for TA glucosylation.
Keywords: Oryza sativa; Glycine max; Solanum tuberosum; Solanum lycopersicum; Nicotiana tabacum; Zea mays; Arabidopsis thaliana; Albizzia julibrissin; Salvia officinalis; Tuberonic acid; Tuberonic acid glucoside; Tuberonic acid glucosyltransferase; Jasmonic acid; Wounding stress; Salicylic acid glucosyltransferase;

Occurrence of D-serine in rice and characterization of rice serine racemase by Yoshitaka Gogami; Katsuyoshi Ito; Yuji Kamitani; Yuki Matsushima; Tadao Oikawa (380-387).
Germinated, unpolished rice (Oryza sativa L.) was found to contain a substantial amount of D-serine, and the ratio of the D-enantiomer to the L-enantiomer was higher for serine than for other amino acids. A serine racemase (E.C.5.1.1.18) was characterized from the source, whose structure was distorted by addition of Mg2+; this structural change probably regulates competing racemase and dehydratase activities.Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg2+ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg2+, and this structural change probably regulates the two enzymatic activities.
Keywords: Unpolished rice; Oryza sativa L.; Gramineae; D-serine; D-amino acid; Serine racemase; Serine dehydratase; Pyridoxal 5′-phosphate;

Generation of primary amide glucosides from cyanogenic glucosides by Jandirk Sendker; Adolf Nahrstedt (388-393).
The conversion of the cyanogenic glucoside prunasin into the corresponding prunasinamide has been observed in the leaves of Olinia ventosa and other prunasin-containing species only if reactive oxygen species were produced. In vitro, pure prunasin was quickly degraded with H2O2 indicating that the Radziszewski reaction is a feasible mechanism for amide generation.The cyanogenic glucoside-related compound prunasinamide, (2R)-β-d-glucopyranosyloxyacetamide, has been detected in dried, but not in fresh leaves of the prunasin-containing species Olinia ventosa, Prunus laurocerasus, Pteridium aquilinium and Holocalyx balansae. Experiments with leaves of O. ventosa indicated a connection between amide generation and an excessive production of reactive oxygen species. In vitro, the Radziszewski reaction with H2O2 has been performed to yield high amounts of prunasinamide from prunasin. This reaction is suggested to produce primary amides from cyanogenic glycosides in drying and decaying leaves. Two different benzoic acid esters which may be connected to prunasin metabolism were isolated and identified as the main constituents of chlorotic leaves from O. ventosa and P. laurocerasus.
Keywords: Olinia ventosa; Oliniaceae; Prunus laurocerasus; Rosaceae; Pteridium aquilinium; Pteridaceae; Holocalyx balansae; Caesalpiniaceae; Cyanogenic glycosides; Primary amides; Prunasin; Prunasinamide; Senescence; Pseudosenescence; Radziszewski reaction;

The phytopathogenic fungus Alternaria brassicicola: Phytotoxin production and phytoalexin elicitation by M. Soledade C. Pedras; Paulos B. Chumala; Wei Jin; Mohammed S. Islam; Dominic W. Hauck (394-402).
Alternaria brassicicola produces brassicicolin A as the major host-selective toxin.The metabolites and phytotoxins produced by the phytopathogenic fungus Alternaria brassicicola (Schwein.) Wiltshire, as well as the phytoalexins induced in host plants, were investigated. Brassicicolin A emerged as the most selective phytotoxic metabolite produced in liquid cultures of A. brassicicola and spirobrassinin as the major phytoalexin produced in infected leaves of Brassica juncea (whole plants). In detached infected leaves of B. juncea, the main component was N′-acetyl-3-indolylmethanamine, the product of detoxification of the phytoalexin brassinin by A. brassicicola. In addition, the structure elucidation of three hitherto unknown metabolites having a fusicoccane skeleton was carried out and the antifungal activity of several plant defenses against A. brassicicola was determined.
Keywords: Alternaria brassicicola; Brown mustard; Brassica juncea; Canola; Brassica napus; White mustard; Sinapis alba; Brassicaceae; Brassicicolin A; Brassicicene; Brassinin; Cyclobrassinin; Destruxin; Fusicoccane; Host-selective; Phytoalexin; Phytotoxin; Spirobrassinin;

Fractionation of the dichloromethane extract of Hypericum revolutum ssp. revolutum and the hexane extract of Hypericum choisianum led to the isolation of two compounds, 3-hydroxy-1,4,7-trimethoxydibenzofuran (1) and 4-(3-O-3″)-3″-methylbutenyl-6-phenyl-pyran-2-one (2), respectively. The minimum inhibitory concentration (MIC) of (1) against a panel of multidrug- and methicillin-resistant Staphylococcus aureus strains was 256 μg/ml.In a project to isolate and characterise anti-staphylococcal compounds from members of the genus Hypericum, a dibenzofuran and a pyranone were isolated from the dichloromethane and hexane extracts of Hypericum revolutum ssp. revolutum Vahl (Guttiferae) and Hypericum choisianum Wall. ex. N. Robson (Guttiferae), respectively. The structures of these compounds were elucidated by 1- and 2D-NMR spectroscopy and mass spectrometry as 3-hydroxy-1,4,7-trimethoxydibenzofuran (1) and 4-(3-O-3″)-3″-methylbutenyl-6-phenyl-pyran-2-one (2). The metabolites were evaluated against a panel of multidrug-resistant strains of Staphylococcus aureus. Compound 1 exhibited a minimum inhibitory concentration (MIC) of 256 μg/ml, whereas compound 2 was inactive at a concentration of 512 μg/ml.
Keywords: Hypericum revolutum; Hypericum choisianum; Guttiferae; Dibenzofuran; Pyranone; Pyran-2-one; Antibacterial; MRSA; Staphylococcus aureus; MDR;

Aromatase inhibitory, radical scavenging, and antioxidant activities of depsidones and diaryl ethers from the endophytic fungus Corynespora cassiicola L36 by Porntep Chomcheon; Suthep Wiyakrutta; Nongluksna Sriubolmas; Nattaya Ngamrojanavanich; Surapong Kengtong; Chulabhorn Mahidol; Somsak Ruchirawat; Prasat Kittakoop (407-413).
Three compounds, corynesidones A, B, and corynether A were isolated from the fungus Corynespora cassiicola, and they exhibited potent antioxidant activity. Corynesidone A (R1 to R4  = H) was an aromatase inhibitor.Isolation of a broth extract of the endophytic fungus Corynespora cassiicola L36 afforded three compounds, corynesidones A (1) and B (3), and corynether A (5), together with a known diaryl ether 7. Compounds 1, 3, 5, and 7 were relatively non-toxic against cancer cells, and inactive toward normal cell line, MRC-5. Corynesidone B (3) exhibited potent radical scavenging activity in the DPPH assay, whose activity was comparable to ascorbic acid. Based on the ORAC assay, compounds 1, 3, 5, and 7 showed potent antioxidant activity. However, the isolated natural substances and their methylated derivatives (18) neither inhibited superoxide anion radical formation in the XXO assay nor suppressed TPA-induced superoxide anion generation in HL-60 cell line. Corynesidone A (1) inhibited aromatase activity with an IC50 value of 5.30 μM.
Keywords: Corynespora cassiicola; Endophytic fungi; Depsidone; Diaryl ether; Aromatase inhibitor; Radical scavenging; Antioxidant activity; Cytotoxic activity;

Norsesquiterpene hydrocarbon, chemical composition and antimicrobial activity of Rhaponticum carthamoides root essential oil by Jaroslav Havlik; Milos Budesinsky; Pavel Kloucek; Ladislav Kokoska; Irena Valterova; Sona Vasickova; Vaclav Zeleny (414-418).
A detailed analysis of Rhaponticum carthamoides (Willd.) Iljin root essential oil was carried out, resulting in identification of 30 constituents. Structure of the main component was elucidated as 13-norcypera-1(5),11(12)-diene and is reported for first time. The oil was active against some Gram-positive bacteria and yeast.A detailed analysis of Rhaponticum carthamoides (Willd.) Iljin root essential oil was carried out by GC, GC–MS and GC–FTIR techniques. In total, 30 components were identified, accounting for 98.0% of total volatiles. A norsesquiterpene 13-norcypera-1(5),11(12)-diene (22.6%), followed by aplotaxene (21.2%) and cyperene (17.9%), were isolated and their structures confirmed by 1D and 2D-NMR spectra (COSY, ROESY, HSQC, HMBC and INADEQUATE). Selinene type sesquiterpenes and aliphatic hydrocarbons were among minor constituents of the essential oil. The oil exhibited antimicrobial activity against 5 of 9 strains of bacteria and yeast, when tested using broth micro-dilution method. Minimum inhibitory concentrations ranged between 32 and 256 μg/ml.
Keywords: Rhaponticum carthamoides; Essential oil; 13-Norcypera-1(5),11(12)-diene; Aplotaxene; Cyperene; Antimicrobial activity;

Triterpenoids with antimicrobial activity from Drypetes inaequalis by Simon Suh Awanchiri; Hanh Trinh-Van-Dufat; Jovita Chi Shirri; Marlise Diane J. Dongfack; Guy Merlin Nguenang; Sabrina Boutefnouchet; Zacharias T. Fomum; Elisabeth Seguin; Philippe Verite; François Tillequin; Jean Wandji (419-423).
Five antimicrobial triterpenoids belonging to lup-20(29)-ene, olean-12-ene and friedelane type derivatives have been isolated from Drypetes inaequalis.The air-dried stems and ripe fruit of Drypetes inaequalis Hutch. (Euphorbiaceae) were studied. Four triterpene derivatives, characterized as lup-20(29)-en-3β,6α-diol, 3β-acetoxylup-20(29)-en-6α-ol, 3β-caffeoyloxylup-20(29)-en-6α-ol and 28-βd-glucopyranosyl-30-methyl 3β-hydroxyolean-12-en-28,30-dioate along with 10 known compounds were isolated from the whole stems. One triterpene, characterized as 3α-hydroxyfriedelan-25-al along with six known compounds were isolated from the ripe fruit. Their structures were established on the basis of spectroscopic analysis and chemical evidence. The triterpenes were tested for antimicrobial activity against some Gram-positive and Gram-negative bacteria, and two of them appeared to be modestly active.
Keywords: Drypetes inaequalis; Euphorbiaceae; Stems; Fruit; Triterpenoid esters; Saponins; Antimicrobial activity;

Seco-tabersonine alkaloids from Tabernaemontana corymbosa by Kuan-Hon Lim; Noel F. Thomas; Zanariah Abdullah; Toh-Seok Kam (424-429).
Two Seco-tabersonine alkaloids jerantiphyllines A and B, in addition to a tabersonine hydroxyindolenine jerantinine H, and a recently reported vincamine alkaloid 7 were isolated from the leaf extract of the Malayan Tabernaemontana corymbosa.Two seco-tabersonine alkaloids, jerantiphyllines A and B, in addition to a tabersonine hydroxyindolenine, jerantinine H, and a recently reported vincamine alkaloid 7, were isolated from the leaf extract of the Malayan Tabernaemontana corymbosa and the structures were established using NMR and MS analysis. Biomimetic conversion of jerantinines A and E to their respective vincamine and 16-epivincamine derivatives were also carried out.
Keywords: Alkaloids; Tabernaemontana; NMR; Plants;

Ecdysteroids and a sucrose phenylpropanoid ester from Froelichia floridana by Ping Wang; Shiyou Li; Stacy Ownby; Zhizhen Zhang; Wei Yuan; Wanli Zhang; R. Scott Beasley (430-436).
Phytoecdysteroid glycosides 15 and a phenylpropanoid ester of sucrose 6 were isolated from Froelichia floridana, along with eight known compounds (714). Compounds 12 and 614 were tested in vitro for their activity against DNA topoisomerase I, but only diosmetin (13) exhibited marginal inhibition.Phytoecdysteroid glycosides (15) and a phenylpropanoid ester of sucrose (6) were isolated from the whole plant of Froelichia floridana , along with eight known compounds including three ecdysteroids (79), four flavonoids (1013), and one phenolic compound (14). Structures were determined using a combination of spectroscopic techniques. Compounds 1, 2 and 614 were tested in vitro for their activity against human DNA topoisomerase I. Compound 13 (diosmetin) showed marginal inhibition against topoisomerase I with IC50 of 130 μM in conjunction with low intercalation ability.
Keywords: Froelichia floridana; Amaranthaceae; Ecdysteroid glycosides; Phytoecdysteroids; Sucrose phenylpropanoid ester; DNA topoisomerase I;

Erratum to “Phytotoxic polyacetylenes from roots of Russian knapweed (Acroptilon repens (L.) DC.)” [Phytochemistry 69 (2008) 2572–2578] by Naira Quintana; Tiffany L. Weir; Jiang Du; Corey D. Broeckling; Julie P. Rieder; Frank R. Stermitz; Mark W. Paschke; Jorge M. Vivanco (437).