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

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 ERα and ERβ, 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.

Urinary Equol and Equol‐Predicting Microbial Species Are Favorably Associated With Cardiometabolic Risk Markers in Chinese Adults

“…First, as a polyphenol, the hydroxyl group in equol can serve as a hydrogen donor for free radicals, thereby reducing the number of free radicals. 32 Second, equol can exert estrogenic or antiestrogenic effects by binding to the estrogen receptor (estrogen receptors ER‐α and ER‐β) 33 and indirectly regulate glucose and lipid metabolism through lipogenesis, lipolysis, and adipogenesis. Ricketts et al found that equol also works by activating peroxisome proliferator‐activaed receptor‐α, which leads to decreased triglyceride concentrations by increasing fatty acid oxidation in the liver or skeletal muscle.…”

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives

“…Mushroom tyrosinase was found to convert equol to 3 ′ -hydroxyequol in the study of the inhibitory effect of equol [77]. EcHpaB and the variant T292A were proved to hydroxylate the C6 position of equol to 6-hydroxyequol [107]. Both HpaB ro-3 derived from Rhodococcus opacus B4 and HpaB pl-1 derived from Photorhabdus luminescens can hydroxylate equol, but at different hydroxylation sites [78,108].…”