ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and a 3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea

ent-Eudesmane sesquiterpenoids, 8,11-dihydroxy-2,4-cycloeudesmane, 11-hydroxy-2,4-cycloeudesman-8-one and 2,4-cyclo-7(11)-eudesmen-8-one, were isolated from the wood of Platycarya strobilacea, which has been used as an aromatic tree since at least the 18th century. On charring the wood, 2,4-cyclo-7(11)-eudesmen-8-one was detected in the smoke. In the charred wood, the concentrations of ellagitannins, such as galloyl pedunculagin, dramatically decreased, whereas concentrations of pentagalloyl glucose, and other gallotannins were relatively stable. In addition, two other compounds, the 6′-O-m- and p-digalloyl oak lactone precursor and the 3-O-methylellagic acid 4′-O-(4″-O-galloyl)-xylopyranoside, were isolated from the charred wood along with m- and p-digallic acid.     

Spatial distributions of the leafminer Ophiomyia maura (Diptera: Agromyzidae) in host plant Aster ageratoides

The seasonal occurrence and among-plant and within-plant spatial distribution of the multivoltine leafminer Ophiomyia maura Meigen (Diptera: Agromyzidae) on the herbaceous plant Aster ageratoides Turcz. subsp. ovatus (Asteraceae) were investigated in the field. O. maura has at least four generations a year and mines per leaf fluctuate with a mean of 0.007 throughout the occurrence period. Seasonal occurrence is associated with abundance of new host leaves, suggesting O. maura females prefer to oviposit in newly emerged leaves. The among-plant distribution of O. maura is described by a Poisson distribution early in the season but tends to be weakly clumped later. The within-plant vertical distribution of larval mines increased from middle to upper leaves during plant development, because mined leaves in the middle position early in the season move downward with the emergence of new leaves, shifting mined leaves from the position where O. maura oviposits eggs. Later in the season, mined leaves remain where they are deposited because few new leaves emerge. The spatial distribution of O. maura , resource utilization patterns, and host plant characteristics are discussed.     

NAD(P)H-dependent 6'-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract

The crude extract prepared from Glycyrrhiza echinata cells treated with yeast extract catalyzed the formation of liquiritigenin (5-deoxyflavanone) and isoliquiritigenin (6'-deoxychalcone) in addition to naringenin (5-hydroxyflavanone) when incubated with 4-coumaroyl-CoA and malonyl-CoA in the presence of high concentrations (0.1 mM or higher) of NADPH. Incubation without NADPH, or with low concentrations (0.01 mM or lower), gave only naringenin as a reaction product. With NADH (1 mM), the major product was naringenin accompanied by a small quantity of liquiritigenin. The initial product of the assay with 1 mM NADPH was isoliquiritigenin, indicating a reaction catalyzed by 6'-deoxychalcone synthase (DOCS). Subsequent formation of liquiritigenin was attributed to the presence of chalcone isomerase in the crude extract. The results constitute the first demonstration in vitro of DOCS activity which, in G. echinata cells and other leguminous plants, is involved in the biosynthesis of retrochalcone and 5-deoxyisoflavonoid-derived phytoalexins.     

Father-to-Mother-to-Infant Transmission of HIV-1: Clonally Transmitted Isolate of Infant Mutates More Rapidly than That of the Mother and Rapidly Loses Reactivity with Neutralizing Antibody

The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type-1 from a father-to-mother-to-infant trimmer were studied and the horizontal and vertical transmissions compared. The father's virus was variable for reactivity with neutralizing antibody and sequences of the V3 loop central core sequence. In contrast, the mother's viral sequences were much less diverse and reacted with a virus neutralizing antibody. The infant's viral sequences were also less diverse than those of the father, and N-glycosylation sites were conserved. By phylogenetic analysis, the major clone, of which V3-peptide reacted with the neutralizing antibody, was found to be transmitted from the mother to her infant; however, the mutated minor clones did not bind to the antibody. These findings suggest that both horizontal and vertical virus transmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus was horizontally transmitted.     

Molecular Cloning and Characterization of a cDNA for Pterocarpan 4-Dimethylallyltransferase Catalyzing the Key Prenylation Step in the Biosynthesis of Glyceollin,a Soybean Phytoalexin

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C₅ prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(−)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-¹³C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.Typical phytoalexins of the Leguminosae are isoflavonoid derivatives with characteristic species-specific modifications in both their skeletons and their decoration, e.g. prenylation (Dixon, 1999). Isoflavonoids are formed through an early branching pathway in flavonoid metabolism. The most abundantly found isoflavonoid skeleton of leguminous phytoalexins is pterocarpan, and more than one-half of these pterocarpanoids are decorated in a complex manner mainly by isoprenoid-derived substituents (Tahara and Ibrahim, 1995). Glyceollin is the collective name for soybean (Glycine max) phytoalexins with pterocarpanoid skeletons and cyclic ether decoration originating from C₅ prenyl substitutions (Fig. 1). The biosynthesis mechanism of soybean phytoalexins has been studied extensively during the 1970s to 1990s, most actively by Grisebach et al. (Ebel and Grisebach, 1988), and the pathway and biosynthetic enzymes involved have been characterized intensively at the biochemical level (Ebel, 1986; Dixon, 1999). More recent studies with leguminous plants such as alfalfa (Medicago sativa), licorice (Glycyrrhiza echinata), Lotus japonicus, and Medicago truncatula in addition to soybean have resulted in the identification of many genes encoding enzymes involved in isoflavonoid formation (Dixon, 1999; Shimada et al., 2007; Veitch, 2007). However, some genes encoding enzymes of the later stages of glyceollin biosynthesis, especially the crucial prenylation step, have remained uncharacterized until now.Open in a separate windowFigure 1.Biosynthesis of glyceollin isomers in soybean. Abbreviations not defined in the text: HID, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; P6aH, pterocarpan 6a-hydroxylase; G2DT, dimethylallyl diphosphate: (−)-glycinol 2-dimethylallyltransferase.During glyceollin biosynthesis, a dimethylallyl group is introduced at either C-4 or C-2 of the pterocarpan skeleton (C-8 or C-6 by isoflavone numbering, respectively). A prenyltransferase activity catalyzing the dimethylallylation of (6aS, 11aS)-3,9,6a-trihydroxypterocarpan, (−)-glycinol, has been demonstrated in microsomal fractions of soybean cotyledons and cell cultures treated with a glucan elicitor derived from the cell walls of Phytophthora sojae (Zähringer et al., 1979). An increased toxicity of the prenylated pterocarpans against a phytopathogenic fungus was also demonstrated (Zähringer et al., 1981). An important finding was that the prenylation activity was localized to the chloroplast fraction of cotyledon cells in contrast to the endoplasmic reticulum (ER) where many of the cytochrome P450s (P450s) for glyceollin formation are localized (Welle and Grisebach, 1988; Biggs et al., 1990; Ayabe and Akashi, 2006). Efficient solubilization of the activity and partial purification of the enzyme have also been reported (Welle and Grisebach, 1991), but no complete purification was achieved to sequence the amino acids, and thus the gene responsible remains unidentified.Recently, plant cDNAs of aromatic substrate prenyltransferases have been characterized, and their nucleotide sequence information has become available (Yazaki et al., 2002; Sasaki et al., 2008). In view of the potential benefits of understanding the molecular mechanism underlying the phytopathogen resistance of soybean for the future disease-resistance breeding, studies toward the complete identification of the enzymes involved in glyceollin biosynthesis are important. Thus, this study undertook the molecular cloning and biochemical characterization of a soybean prenyltransferase involved in the glyceollin biosynthetic pathway.     

A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicus

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6'-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6'-deoxychalcone and 6'-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1-cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1-3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.     

Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase

Isoflavonoids are distributed predominantly in leguminous plants, and play pivotal roles in the interaction of host plants with biological environments. Isoflavones in the diet also have beneficial effects on human health as phytoestrogens. The isoflavonoid skeleton is constructed by the CYP93C subfamily of cytochrome P450s in plant cells. The reaction consists of hydroxylation of the flavanone molecule at C-2 and an intramolecular 1,2-aryl migration from C-2 to C-3 to yield 2-hydroxyisoflavanone. In this study, with the aid of alignment of amino acid sequences of CYP93 family P450s and a computer-generated putative stereo structure of the protein, candidates for key amino acid residues in CYP93C2 responsible for the unique aryl migration in 2-hydroxyisoflavanone synthase reaction were identified. Microsomes of recombinant yeast cells expressing mutant proteins of CYP93C2 were prepared, and their catalytic activities tested. The reaction with the mutant in which Ser 310 in the centre of the I-helix was converted to Thr yielded increased formation of 3-hydroxyflavanone, a by-product of the 2-hydroxyisoflavanone synthase reaction, in addition to the major isoflavonoid product. More dramatically, the mutant in which Lys 375 in the end of beta-sheet 1-4 was replaced with Thr produced only 3-hydroxyflavanone and did not yield the isoflavonoid any longer. The roles of these amino acid residues in the catalysis and evolution of isoflavonoid biosynthesis are discussed.     

The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.     

New scheme of the biosynthesis of formononetin involving 2,7,4'-trihydroxyisoflavanone but not daidzein as the methyl acceptor

Glycyrrhiza echinata cell-free extract produced isoformononetin by the 7-O-transmethylation of daidzein from S-adenosyl-L-methionine (SAM). When the yeast microsome expressing 2-hydroxyisoflavanone synthase was mixed with the cell-free extract and incubated with liquiritigenin and SAM, formononetin emerged. Furthermore, the cell-free extract yielded formononetin on incubation with 2,7,4'-trihydroxyisoflavanone and SAM. We propose a novel pathway of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor.