title: Regioselectivity-driven evolution of CYP102D1 for improved synthesis of 3'-ortho-dihydroxyisoflavone.
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.
authors.affiliation: Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, South Korea. Electronic address: kychoi@ajou.ac.kr.
authors.forename: Kwon-Young
authors.initials: KY
authors.lastname: Choi
authors.affiliation: Department of Microbial Engineering, College of Engineering, Konkuk University, South Korea.
authors.forename: Yung-Hun
authors.initials: YH
authors.lastname: Yang
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D057166:High-Throughput Screening Assays; D006900:Hydroxylation; D007529:Isoflavones / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007529:Isoflavones; D011994:Recombinant Proteins; C004742:daidzein; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: Cytochrome P450 monooxygenases; Daidzein; O-dealkylation HTS assay; Regioselectivity; Self-sufficient CYP102D1