Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants

Hanbit Song; Pyung-Gang Lee; Junyeob Kim; Joonwon Kim; Sang-Hyuk Lee; Hyun Kim; Uk-Jae Lee; Jin Young Kim; Eun-Jung Kim; Byung-Gee Kim

doi:10.3389/fbioe.2022.830712

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants

Front Bioeng Biotechnol. 2022 Mar 24:10:830712. doi: 10.3389/fbioe.2022.830712. eCollection 2022.

Authors

Hanbit Song^{1

2}, Pyung-Gang Lee^{1

2

3}, Junyeob Kim^{1

2}, Joonwon Kim^{1

2}, Sang-Hyuk Lee^{1

2}, Hyun Kim^{1

2}, Uk-Jae Lee^{1

2}, Jin Young Kim^{1

2}, Eun-Jung Kim⁴, Byung-Gee Kim^{1

2

4

5}

Affiliations

¹ School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.
² Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
³ Institute of Engineering Research, Seoul National University, Seoul, South Korea.
⁴ Bio-MAX/N-Bio Institute, Seoul National University, Seoul, South Korea.
⁵ Institute for Sustainable Development (ISD), Seoul National University, Seoul, South Korea.

Abstract

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol-converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3'-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3'HE were also prepared from genistein and 3'-hydroxydaidzein (3'HD or 3'-ODI), respectively. In the following efficacy tests, 3'HE and 6HE showed about 30∼200-fold higher EC₅₀ than (S)-equol in both ER_α and ER_β, and they did not have significant SERM efficacy except 6HE showing 10% lower β/α ratio response than that of 17β-estradiol. In DPPH radical scavenging assay, 3'HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3'HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.

Keywords: SERM (selective estrogen receptor modulator); antioxidants; enzyme engineering; hydroxy-(S)-equol; isoflavonoids; one-pot synthesis; oxidoreductases.