title: Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.).
abstract: Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Waki
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
authors.forename: DongChan
authors.initials: D
authors.lastname: Yoo
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Fujino
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Mameda
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan; Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Denessiouk
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Department of Biological and Environmental Science, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Motohashi
authors.affiliation: Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Akashi
authors.affiliation: Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Aoki
authors.affiliation: Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Ayabe
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan. Electronic address: nakayama@seika.che.tohoku.ac.jp.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005419:Flavonoids / Q000378:metabolism*; D019753:Intramolecular Lyases / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology*
chemical_list: D005419:Flavonoids; D011994:Recombinant Proteins; C402170:cytochrome P-450 CYP93C2 (Glycyrrhiza echinata); D003577:Cytochrome P-450 Enzyme System; D000217:Acyltransferases; C020141:flavanone synthetase; D019753:Intramolecular Lyases; C019865:chalcone isomerase
keywords: 2-Hydroxyisoflavanone synthase; Chalcone isomerase; Chalcone synthase; Isoflavone; Metabolon; Protein–protein interaction