Identification of a Novel Dihydrodaidzein Racemase Essential for Biosynthesis of Equol from Daidzein in Lactococcus sp. Strain 20-92

Abstract

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...

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

“…The unusually large difference in their retention times arose from use of a different chiral column (Chiralcel OJ-H column) under normal-phase conditions, whereas most other researchers have utilized other chiral columns under reversed-phase conditions. It has been claimed that (S)-DHD is eluted first, followed by (R)-DHD (9,20). Additionally, comparison with the retention times of (R)-DHD produced by purified DZNR and of (S)-DHD racemized from (R)-DHD by purified DDRC confirmed the assignment.…”

“…However, the first gene identification for equol metabolism was reported for the non-Coriobacteriaceae microbe Lactococcus sp. strain 20-92 (9)(10)(11). Similar identifications of equol metabolism in Slackia sp.…”

“…1): daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR), and dihydrodaidzein racemase (DDRC). DZNR reduces daidzein to (R)-dihydrodaidzein [(R)-DHD] enantioselectively (9,11). (R)-DHD is subsequently racemized to (S)-DHD either through tautomerization at C-3 or by DDRC activity (9).…”

“…DZNR reduces daidzein to (R)-dihydrodaidzein [(R)-DHD] enantioselectively (9,11). (R)-DHD is subsequently racemized to (S)-DHD either through tautomerization at C-3 or by DDRC activity (9). DHDR is involved in the reduction of (S)-and (R)-DHD to trans-and cis-tetrahydrodaidzein (trans-and cis-THD), respectively (10).…”

Absolute Configuration of (-)-2-(4-Hydroxyphenyl)propionic acid: Stereochemistry of Soy Isoflavone Metabolism

“…In details, genistein is stereospecifically reduced to (R)-dihydrogenistein, which is further isomerized to (S)-dihydrogenistein by enzymatic or abiological keto-enol tautomerization, similarly to daidzein metabolism. 14,23 Further sequential reduction of (S)-dihydrogenistein, via (3S,4R)-tetrahydrogenistein, is believed to produce (S)-5-hydroxyequol. Alternative metabolism of (S)-dihydrogenistein is known to produce 6'-hydroxy-O-DMA and which further metabolized to phloroglucinol and (R)-2-HPPA.…”