Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H

Abstract

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or 'metabolons', which represent the molecular level of organization. Here, we have confirmed the existence of the long-postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4-hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non-consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.

Transcriptomic evidence for the control of soybean root isoflavonoid content by regulation of overlapping phenylpropanoid pathways

“…Overrepresentation and pathway enrichment statistics (Additional file 11: Tables S6, Additional file 12: Tables S7, Additional file 13: Tables S8, and Additional file 14: Tables S9) show that genes involved in secondary metabolism, particularly those encoding hydrolase and oxidoreductase activity are significantly enriched in the ‘low isoflavonoid’ gene list. Membrane integral proteins are also overrepresented within this list, which could impact the formation of isoflavonoid or flavonoid multi-enzyme complexes or ‘metabolons’ [8, 55–57]. …”

“…GmCHS7 and GmCHS8 are critical for isoflavonoid biosynthesis and accumulation in soybean seeds [7]. These two enzymes have shown differential localization; GmCHS7 was localized to the cytoplasm, while GmCHS8 was localized to the nucleus and cytoplasm [8]. The level of differentiation in the expression of almost identical genes in the CHS family, and the putative functional specialization of their cognate proteins underlines the complexity associated with multi-gene families in large genomes such as soybean.…”

Membrane‐Mediated Protein–Protein Interactions of Cholesterol Side‐Chain Cleavage Cytochrome P450 with its Associated Electron Transport Proteins

“…Overall, this study suggests that P450scc and its electron transfer proteins act like a metabolon and that binding of components to a lipid membrane is an important part of this metabolon. Metabolons are temporary complexes of proteins involved in a sequential enzymatic pathway . The first step in the steroid biosynthetic pathway requires P450scc to cleave the side chain of cholesterol.…”

“…Overall, this study suggests that P450scc and its electron transfer proteins act like ametabolon andthat binding of components to al ipid membrane is an important parto ft his metabolon.M etabolons are temporary complexes of proteins involved in as equential enzymatic pathway. [51][52][53] The first step in the steroidb iosynthetic pathway requires P450scc to cleave the side chain of cholesterol.U nlike the bacterial cousins of the mitochondrial( class I) P450s,t he QCM data support ar ole for the membrane in providing at wo-dimensional surface to concentrate the metabolon of P450scc. This is not an ew concept, as Srere and Sumegi in 1986 [54] noted that the mitochondrial matrix is ac rowdedm acromolecular environment.…”

Isoflavonoid‐specific prenyltransferase gene family in soybean: GmPT01, a pterocarpan 2‐dimethylallyltransferase involved in glyceollin biosynthesis

“…Soybean is a paleopolyploid whose genome has undergone two whole genome duplications at approximately 59 and then 13 million years ago, resulting in 75% of genes within the genome in multiple copies (Schmutz et al, 2010). Many isoflavonoid biosynthetic genes contain large gene families where spatio-temporal expression and subcellular location of specific members of the gene family determine the composition of the isoflavonoid metabolon and synthesis of specific metabolites (Dastmalchi et al, 2016). Therefore, it is crucial to identify all the members of the gene families involved in the glyceollin biosynthetic pathway, and determine their role at diverse circumstances.…”