Cloning and expression of a novel NADP(H)-dependent daidzein reductase, an enzyme involved in the metabolism of daidzein, from equol-producing Lactococcus strain 20-92.
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 purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. 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.
Shimada
Yoshikazu
Y
Institute of Biomedical Innovation, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan. yshimada@research.otsuka.co.jp
Yasuda
Setsuko
S
Takahashi
Masayuki
M
Hayashi
Takashi
T
Miyazawa
Norihiro
N
Sato
Ikutaro
I
Abiru
Yasuhiro
Y
Uchiyama
Shigeto
S
Hishigaki
Haretsugu
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D060754:Equol; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007529:Isoflavones / Q000378:metabolism*; D017045:Lactococcus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
C007610:4',7-dihydroxy-3,4-dihydroisoflavone; D003067:Coenzymes; D007529:Isoflavones; D011994:Recombinant Proteins; C433163:dihydrodaidzein; D060754:Equol; C004742:daidzein; D009247:NADH, NADPH Oxidoreductases