Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol inPueraria mirifica

Author:

Suntichaikamolkul Nithiwat1,Akashi Tomoyoshi2,Mahalapbutr Panupong3,Sanachai Kamonpan4,Rungrotmongkol Thanyada56,Bassard Jean-Etienne7ORCID,Schaller Hubert7,De-Eknamkul Wanchai8ORCID,Vimolmangkang Sornkanok89,Yamazaki Mami1011,Sirikantaramas Supaart1ORCID

Affiliation:

1. Center of Excellence in Molecular Crop, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Pathumwan , Bangkok 10330, Thailand

2. Department of Applied Biological Sciences, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa , Kanagawa 252-0880, Japan

3. Department of Biochemistry, Faculty of Medicine, Khon Kaen University , 123 Muang District, Khon Kaen 40002, Thailand

4. Department of Biochemistry, Faculty of Science, Khon Kaen University , 123 Muang District, Khon Kaen 40002, Thailand

5. Center of Excellence in Biocatalyst and Sustainable Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Pathumwan , Bangkok 10330, Thailand

6. Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University , 254 Pathumwan, Bangkok 10330, Thailand

7. Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg , 12 rue du général Zimmer, Strasbourg 67084, France

8. Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Pathumwan , Bangkok 10330, Thailand

9. Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, 254 Pathumwan , Bangkok 10330, Thailand

10. Laboratory of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Science, Chiba University, 1-8-1 Inohana, Chuo-ku , Chiba 260-8675, Japan

11. Plant Molecular Science Center, Chiba University, 1-8-1 Inohana, Chuo-ku Chiba 260-8675, Japan

Abstract

AbstractWhite Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2′ or 3′ daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Funder

Franco-Thai Mobility Programme

Chulalongkorn University

Publisher

Oxford University Press (OUP)

Subject

Cell Biology,Plant Science,Physiology,General Medicine

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