Identification of protein–protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.)

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

“…In the A. thaliana flavonoid metabolon, CHS has structural and catalytic roles, functioning as a hub for the formation of the metabolon and synthesizing the flavonoid precursor (THC), respectively (Winkel‐Shirley, ; Crosby et al ., ). Recent reports described a metabolon for isoflavone biosynthesis in soybean (a Fabales plant), where flavonoid enzymes are tethered to the ER via two P450 proteins (2‐hydroxyisoflavanone synthase and cinnamate 4‐hydroxylase; Dastmalchi et al ., ; Waki et al ., ). In this case, the activities of these P450s were indispensable for isoflavone biosynthesis.…”

“…The S. cerevisiae strain NMY51 [ MAT α his3 Δ 200 trp1‐901 leu2‐3,112 ade2 LYS2::(lexAop)4‐HIS3 ura3::(lexAop)8‐lacZ ade2::(lexAop)8‐ADE2 GAL4 ], which was included in the DUAL membrane Kit 3, was transformed with one of the following pairs of plasmids [derivatives of pBT3‐SUC and pPR3‐N; derivatives of pBT3‐SUC and pPR3‐C; or, as a positive control, pOst1‐NubI (plasmid expressing a fusion protein comprising the yeast resident ER protein Ost1 and the wild‐type Nub portion of yeast ubiquitin) and derivatives of pBT3‐SUC] using the polyethylene glycol–lithium acetate method described in the manufacturer's instructions. Interactions between the tested proteins were also determined based on the growth and β‐galactosidase activities of the transformed cells (Waki et al ., ). Specifically, a single colony on synthetic dropout (SD) agar medium lacking tryptophan and leucine (SD/−WL) was transferred to liquid SD/−WL medium, which was then incubated at 30°C overnight with shaking.…”

“…Yeast two‐hybrid assays were completed using the Matchmaker Gold Yeast Two‐Hybrid System (Clontech, Mountain View, CA, USA) and a slightly modified version of a published procedure (Waki et al ., ). Specifically, S. cerevisiae strain AH109 (Clontech) was transformed with each pair of plasmids encoding proteins to be tested for a possible interaction.…”

“…Two recent studies on Glycine max , which characteristically accumulates isoflavones, confirmed that P450 enzymes (i.e. 2‐hydroxyisoflavanone synthase and cinnamate 4‐hydroxylase) are important metabolon components for isoflavone biosynthesis (Dastmalchi et al ., ; Waki et al ., ). These findings provide further evidence that P450 proteins serve as metabolon components related to plant specialized metabolism.…”

Identification and characterization of a major QTL underlying soybean isoflavone malonylglycitin content

“…Enzymes related to the biosynthesis of isoflavones are considered to be involved in metabolon that perform sequential reactions (Laursen et al 2015). Isoflavone synthase (IFS, a membrane-bound P450 protein) is anchored to the endoplasmic reticulum mem brane, and some related cytoplasmic enzymes, such as chal cone isomerase, CHS, and IFS, are physically associated with each other (Waki et al 2016). The affinity of proteinprotein interactions and the efficient transfer of substrates between enzymes, however, are affected by the combination…”