Molecular characterization and functional expression of dihydroxypterocarpan 6a‐hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (<i>Glycine max</i> L.)

Schopfer, Christel R; Kochs, Georg; Lottspeich, Friedrich; Ebel, Jürgen

doi:10.1016/s0014-5793(98)00866-7

Cited by 67 publications

(44 citation statements)

References 18 publications

(36 reference statements)

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“…In glyceollin I biosynthesis, the direct flavonoid substrate of G4DT is produced at the ER by the P450 3,9-dihydroxypterocarpan 6a-hydroxylase (Schopfer et al, 1998), and the product of G4DT supplies the substrate of another P450 enzyme, a cyclase, in the ER again (Welle and Grisebach, 1988). Although the physiological meaning of the intracellular movement of biosynthetic intermediates is unknown, the present study may enable the characterization the P450 cyclase at the molecular level by providing the appropriate substrate.…”

Section: Plastid Localization Of Flavonoid Prenyltransferasesmentioning

confidence: 87%

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

et al. 2008

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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.

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Section: Plastid Localization Of Flavonoid Prenyltransferasesmentioning

confidence: 87%

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

et al. 2008

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“…G4DT was shown to localize at the plastids (Akashi et al 2009), similar to other flavonoid prenyltransferases identified to date ). However, glycinol is biosynthesized by 3,9-dihydroxypterocarpan 6a-hydroxylase, a cytochrome P450 localized at the ER (Schopfer et al 1998), suggesting an as yet uncharacterized mechanism of glycinol transport into the plastids. Moreover, 4-dimethylallylglycinol, the product of G4DT, is cyclized by another cytochrome P450 localized at the ER (Welle and Grisebach 1988).…”

Section: Interactions With Pathogensmentioning

confidence: 99%

Flavonoids in plant rhizospheres: secretion, fate and their effects on biological communication

Sugiyama

Yazaki

2014

Plant Biotechnology

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Flavonoids, one of the most-described group of plant "specialized metabolites", consist of more than 10,000 structurally diverse compounds. Most flavonoids accumulate in plant vacuoles as glycosides, with some released by the roots into rhizospheres. These flavonoids are involved in biological communications with rhizobia, arbuscular mycorrhizal fungi, plant growth promoting rhizobacteria, pathogens, nematodes, and other plant species. Both aglycones and glycosides of flavonoids are found in root exudates and in soils. This review describes researches on the mechanisms of flavonoid secretion and the fate of flavonoids released into rhizospheres. This review also discusses the direction of future research that may elucidate the specific roles of flavonoids in biological communications in rhizospheres, enabling the utilization of flavonoid activities and functions in agricultural practice.

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“…Previous analyses of the catalytic properties of recombinant proteins expressed in yeast disclosed the function of two of the clones. CYP73A11 cDNA encoded a cinnamate 4-hydroxylase (17), whereas CYP93A1 cDNA coded for 3,9-dihydroxypterocarpan 6a-hydroxylase (18). The former cytochrome P-450 thus represented a well studied enzyme of general phenylpropanoid metabolism whose action gives rise to the hydroxyl group in position 4Ј of the flavonoid B-ring.…”

Section: Flavonoid 6-hydroxylase Cyp71d9 From Soybeanmentioning

confidence: 99%

“…Eight full-length cDNA clones were subsequently isolated that represented elicitor-activated cytochromes P-450. One of these, CYP73A11 cDNA, encoded cin-namate 4-hydroxylase, and a second one, CYP93A1 cDNA, coded for 3,9-dihydroxypterocarpan 6a-hydroxylase (17,18).…”

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confidence: 99%

Flavonoid 6-Hydroxylase from Soybean (Glycine maxL.), a Novel Plant P-450 Monooxygenase

Latunde-Dada¹,

Cabello‐Hurtado²,

Czittrich³

et al. 2001

Journal of Biological Chemistry

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114

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Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 M) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol-and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.Flavonoids are a diverse group of natural products that serve important roles in plants during growth, during development, and in defense against microorganisms and pests (1, 2). These compounds are synthesized from phenylpropanoid-and acetate-derived precursors through central pathways furnishing basic C 6 -C 3 -C 6 flavonoid skeletons and, in addition, through a variety of reactions leading to a range of modified aglycones and subsequently to their glycosylated derivatives within each flavonoid class. Many of the enzymes of flavonoid biosynthesis have been extensively studied (3), and recent molecular biological approaches have complemented biochemical methods in elucidating the mechanism and regulation of flavonoid biosynthesis (4). Typical enzymes belonging to the complex branch pathways for the elaboration of flavonoid skeletons are cytochrome P-450-dependent hydroxylases (3), such as flavonoid 3Ј-hydroxylase (5), flavonoid 3Ј,5Ј-hydroxylase (6), isoflavone 2Ј-hydroxylase (7), flavanone 2-hydroxylase (8), flavone synthase II (9, 10), and 2-hydroxyisoflavanone synthase (2HIS) 1 (11-13). Whereas flavonoid 3Ј-hydroxylase and flavonoid 3Ј,5Ј-hydroxylase are responsible for the formation of the 3Ј,5Ј-hydroxylation pattern of the flavonoid B-ring, hydroxylation of the isoflavone B-ring at the 2Ј position (isoflavone 2Ј-hydroxylase) is one of the key reactions leading to pterocarpan structures. The formation of flavones and isoflavones from flavanones is catalyzed by several evolutionarily related P-450s, either in a ...

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Molecular characterization and functional expression of dihydroxypterocarpan 6a‐hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.)

Cited by 67 publications

References 18 publications

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

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

Flavonoids in plant rhizospheres: secretion, fate and their effects on biological communication

Flavonoid 6-Hydroxylase from Soybean (Glycine maxL.), a Novel Plant P-450 Monooxygenase

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