Related Experiment Video
Updated: Apr 16, 2026

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade
Published on: August 14, 2019
Regioselectivity-driven evolution of CYP102D1 for improved synthesis of 3'-ortho-dihydroxyisoflavone
Kwon-Young Choi1, Yung-Hun Yang2, Byung-Gee Kim3
1Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, South Korea.
Abstract:
Daidzein is a major component of isoflavones, and its hydroxylated forms are valuable phytochemicals with anti-cancer and anti-oxidant activity. Due to the limitations of chemical synthesis of these hydroxylated structures, alternative enzymatic synthesis has been attempted. Previously, several protein-engineering approaches using CYP102D1 were investigated; these produced mutants with daidzein hydroxylation activity and regioselectivity through rational design (F96V/M246I) and saturation mutagenesis (A273H/G274E/T277G). However, the generated mutants have low regioselectivity (F96V/M246I) or low hydroxylation activity (A273H/G274E/T277G). Here, we characterized mutants capable of catalyzing C3'-specific daidzein hydroxylation with enhanced hydroxylation activity and regioselectivity. In order to obtain regioselectivity toward the daidzein C3'-position, site-saturation mutagenesis on the substrate-binding region of CYP102D1 F96V/M246I was investigated. A high-throughput screening assay was then performed, based on O-dealkylation activity against the daidzein analog substrate 4'-O-methyl-daidzein. This resulted in a mutant with more than 23-fold improved hydroxylation activity (55.6±17.9μM(-1)min(-1), or 48.4mg/L titer) and regioselectivity over the 3'/6-position that was increased by three-fold (from 0.9 to 2.6) compared with the F96V/M246I template enzyme. Furthermore, we carried out docking simulation studies that could partially explain the effects of these mutations on C3'-specific hydroxylation activity.
Related Concept Videos
Pharmacogenetics of Phase I Enzymes: Cytochrome P450 Isozymes
Regioselectivity and Stereochemistry of Acid-Catalyzed Hydration
Regioselective Formation of Enolates
Regioselectivity of Electrophilic Additions-Peroxide Effect
Regioselectivity and Stereochemistry of Hydroboration
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn...
[3,3] Sigmatropic Rearrangement of 1,5-Dienes: Cope Rearrangement

