Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli.The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.Isoflavones are flavonoids present in various plants and are known to be abundant in soybeans and legumes. These compounds have been called phytoestrogens because their chemical structure is similar to that of the female sex hormone, estrogen. Isoflavones have an ability to bind to estrogen receptors and show protection against or improvement in several sex hormone-dependent diseases, such as breast cancer, prostate cancer, menopausal disorder, lower bone density, and hypertension, due to their weak agonistic or antagonistic effects (1,19,27).Daidzein is one of the main soy isoflavonoids produced from daidzin by the glucosidase of intestinal bacteria (17). Equol is a metabolite produced from daidzein by the enterobacterial microflora (5). Recently, equol has attracted a great deal of attention because its estrogenic activity is more potent than that of other isoflavonoids, including daidzein (27). It is well known that individual variation exists in the ability of these enteric microflora to produce equol and that less than half the human population is capable of producing equol after ingesting soy isoflavones (3). Therefore, to increase the production of equol in the enteric environment of each individual, the development of probiotics using safe bacteria which have the ability to produce equol from daidzein is ongoing.Lactococcus strain 20-92 (Lactococcus 20-92; 30a) is an equol-producing lactic acid bacterium isolated from the feces of healthy humans by Uchiyama et al. (30). This bacterium is spherical and Gram positive and is a strain of L. garvieae. The application of Lactococcus 20-92 in probiotics is advantageous because L. garvieae is not pathogenic or toxic to humans.To date, other bacterial strains that are capable of transforming daidzein to dihydrodaidzein or equol have been isolated (9,21,22,23,29,32,36,37). Daidzein is thought to be metabolized by human intestinal bacteria to equol or to Odesmethylangolensin via dihydrodaidzein and tetrahydrodaidzein (14,15,22,32); however, neither the enzymes involved in the metabolism of daidzein to equol nor even the metabolic pathway has been clarified fully for equol-producing bacteria.In this study, we purified an enzyme from Lactococcus 20-92 that assisted in the conversion of daidzein to dihydrodaidzein. Furthermore, we cloned the...

Equol is metabolized from daidzein, a soy isoflavone, by the gut microflora. In this study, we identified a novel dihydrodaidzein racemase (L-DDRC) that is involved in equol biosynthesis in a lactic acid bacterium, Lactococcus sp. strain 20-92, and confirmed that histidine-tagged recombinant L-DDRC (L-DDRC-His) was able to convert both the (R)-and (S)-enantiomers of dihydrodaidzein to the racemate. Moreover, we showed that recombinant L-DDRC-His was essential for in vitro equol production from daidzein by a recombinant enzyme mixture and that efficient in vitro equol production from daidzein was possible using at least four enzymes, including L-DDRC. We also proposed a model of the metabolic pathway from daidzein to equol in Lactococcus strain 20-92. The intake of isoflavones through soy foods (e.g., miso, tofu, and natto) has many reported health benefits. Because isoflavones are structurally similar to the female hormone estrogen, the associated health benefits are thought to be due to their abilities to bind to estrogen receptors (9, 10, 13). Among the isoflavonoids, equol, a metabolite of daidzein produced by members of the gut microflora (4), is thought to be the primary soy isoflavone derivative that is responsible for the prevention of several sex hormonedependent diseases because of its potent estrogenic activity (13). However, individual differences exist in the ability of the enteric microflora to produce equol, and equol cannot be produced in more than half of the individuals who consume soy isoflavones (2). Therefore, it has been proposed that the development of probiotics using safe bacteria capable of producing equol from daidzein could allow the production of equol in the enteric environments of all individuals.Lactococcus sp. strain 20-92, isolated from healthy human feces, is an equol-producing lactic acid bacterium that produces equol from daidzein and is classified as Lactococcus garvieae, which is found in several traditional Italian cheeses and in healthy human intestines (6, 16). The application of Lactococcus strain 20-92 to foods is currently being investigated.Equol is thought to be produced sequentially from daidzein via dihydrodaidzein and tetrahydrodaidzein by intestinal bacteria (7,17). To elucidate the metabolic pathway from daidzein to equol in Lactococcus strain 20-92, we recently identified three enzymes (daidzein reductase [L-DZNR], dihydrodaidzein reductase [L-DHDR], and tetrahydrodaidzein reductase [L-THDR]) that catalyze the successive conversion steps of the metabolic pathway and showed that the genes for these enzymes form a gene cluster in the bacterium's genome (14, 15). Dihydrodaidzein, the first metabolite in the production of equol from daidzein, has two enantiomers, as does equol, because of the presence of an asymmetric carbon atom at the C-3 position. We recently showed that although the dihydrodaidzein that was produced by recombinant L-DZNR was largely (S)-dihydrodaidzein, a racemic mixture of dihydrodaidzein was detected when daidzein was converted using a cell e...

The ING1 gene encodes p33^ING1, a putative tumor suppressor for neuroblastomas and breast cancers, which has been shown to cooperate with p53 in controlling cell proliferation. We have isolated a novel human gene, ING1L, that potentially encodes a PHD-type zinc-finger protein highly homologous to p33^ING1. Fluorescence in situ hybridization and radiation-hybrid analyses assigned ING1L to human chromosome 4. Both ING1 and ING1L are expressed in a variety of human tissues, but we found ING1L expression to be significantly more pronounced in tumors from several colon-cancer patients than in normal colon tissues excised at the same surgical sites. Although the significance of this observation with respect to carcinogenesis remains to be established, the data suggest that ING1L might be involved in colon cancers through interference with signal(s) transmitted through p53 and p33^ING1.

Lactococcus strain 20–92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20–92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20–92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20–92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.

We isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1-q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.