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.

Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4#-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNAwas cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4#-methoxyisoflavanone, while soybean HID has broader specificity to both 4#-hydroxylated and 4#-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.

Flavonoids are common to most vascular plants and are postulated to be involved in a number of plant functions. Leguminous plants are intriguing materials for studies on flavonoids for several reasons. First, legumes produce flavonoids with specific chemical structures i.e. isoflavonoids and 5-deoxy( iso)f lavonoids. Second, leg um i nous flavonoids are postulated to play important roles both as defensive compounds against pathogenic microorganisms and as chemical signals in symbiotic nitrogen fixation. Third, as constituents of food and forage, leguminous flavonoids have striking biological activity in humans and animals. This article reviews the chemical structure, biological activity, and biosynthesis of leguminous flavonoids. Recent achievements in the identification and characterization of cytochrome P450 monooxygenases, which play important roles in (iso) flavonoid biosynthesis, are also introduced. Perspectives of molecular genetic studies on flavonoids with the model legume are discussed.

Isoflavonoids are distributed predominantly in leguminous plants and play critical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisoflavanone synthase, is the key enzyme in their biosynthesis. In cultured licorice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an isoflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin) is rapidly induced upon elicitation. In this study, we obtained a full-length P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. echinata cells. When the flavanones liquiritigenin and naringenin were incubated with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and genistein by acid treatment. The chemical structures of the products from liquiritigenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectrometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, which catalyzes the hydroxylation associated with 1,2-aryl migration of flavanones. Northern-blot analysis revealed that transcripts of CYP93C2, in addition to those of other P450s involved in phenylpropanoid/flavonoid pathways, transiently accumulate upon elicitation.