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

Abstract

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.

Molecular Characterization of a Membrane-bound Prenyltransferase Specific for Isoflavone from Sophora flavescens

“…A phylogenic tree was made with these candidates and flavonoid prenyltransferases previously reported (SfN8DT-1, SfN8DT-2 isolated from S. flavescens, G4DT from Glycine max) (14,15), in addition to homogentisate prenyltransferases involved in vitamin E and plastoquinone biosynthesis (Fig. 2).…”

“…In our recent study, the first cDNA encoding flavanone-specific plant prenyltransferase, naringenin 8-dimethylallyltransferase (SfN8DT-1) from Sophora flavescens, was reported (14). After the discovery of SfN8DT-1, it was expected that prenyltransferases recognizing more divergent substrates such as flavones and isoflavones could be cloned, but thus far only a pterocarpan-specific prenyltransferase has been reported from soybeans (15).…”

The International Conference of Natural Product Biosynthesis (ICNPB, 8th US–Japan seminar on the Biosynthesis of Natural Products)

“…Dr Tomoyoshi Akashi (Nihon University) presented the characterization of a 2-hydroxyisoflavanone synthase in nonleguminous plant and a prenyltransferase responsible for glyceollin and other phytoalexin biosynthesis in soybean. 25,26 Dr Hikaru Seki (Osaka University) reported activities of several P-450 enzymes responsible for sitespecific oxidations of b-amyrin. 27 These oxidations were characterized by heterologous expression in the yeast.…”

Soyabean glyceollins: biological effects and relevance to human health

“…Enzymes involved in glyceollin biosynthesis have been thoroughly characterised during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned. However, some genes need to be identified for later biosynthetic steps (27) . According to a recent study by Akashi et al, dimethylallyl diphosphate: (6aS, 11aS)-3,9, 6a-trihydroxypterocarpan ((2)-glycinol) 4-dimethylallyltransferase appears to be involved in yielding the direct precursor of glyceollin I (27) .…”

“…However, some genes need to be identified for later biosynthetic steps (27) . According to a recent study by Akashi et al, dimethylallyl diphosphate: (6aS, 11aS)-3,9, 6a-trihydroxypterocarpan ((2)-glycinol) 4-dimethylallyltransferase appears to be involved in yielding the direct precursor of glyceollin I (27) . Glyceollins are derived from the precursor molecule, daidzein through several intermediate structures including glycinol (28) .…”