Open AccessIdentification of an Enzyme System for Daidzein-to-Equol Conversion in Slackia sp. Strain NATTS
Author(s) -
Hirokazu Tsuji,
Kaoru Moriyama,
Koji Nomoto,
Hideyuki Akaza
Publication year - 2012
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.06779-11
Subject(s) - open reading frame , daidzein , escherichia coli , biology , reductase , equol , gene , strain (injury) , biochemistry , microbiology and biotechnology , enzyme , peptide sequence , genetics , genistein , anatomy
AnEscherichia coli library comprising 8,424 strains incorporating gene fragments of the equol-producing bacteriumSlackia sp. strain NATTS was constructed and screened forE. coli strains having daidzein- and dihydrodaidzein (DHD)- metabolizing activity. We obtained 3 clones that functioned to convert daidzein to DHD and 2 clones that converted DHD to equol. We then sequenced the gene fragments inserted into plasmids contained by these 5 clones. All of the gene fragments were contiguous, encoding three open reading frames (ORF-1, -2, and -3). Analysis ofE. coli strains containing an expression vector incorporating one of theorf-1 ,-2 , or-3 genes revealed that (i) the protein encoded byorf -1 was involved in the conversion ofcis/trans- tetrahydrodaidzein (cis/trans- THD) to equol, (ii) the protein encoded byorf -2 was involved in the conversion of DHD tocis/trans- THD, and (iii) the protein encoded byorf -3 was involved in the conversion of daidzein to DHD. ORF-1 had a primary amino acid structure similar to that of succinate dehydrogenase. ORF-2 was presumed to be an enzyme belonging to the short-chain dehydrogenase/reductase superfamily. ORF-3 was predicted to have 42% identity to the daidzein reductase ofLactococcus strain 20-92 and belonged to the NADH:flavin oxidoreductase family. These findings showed that the daidzein-to-equol conversion reaction in theSlackia sp. NATTS strain proceeds by the action of these three enzymes.