Role of Sucrose Synthase in the Metabolism of Indican

Abstract

Indigo,a blue dye, is obtained from the cultivated plant species Polygonum tinctorium. It is derived from indican (indoxyl β‐D‐glucoside), one of the secondary metabolites stored in the vacuole of the leaf cells. In the cells, indican is biosynthesized by the catalysis of indoxyl b‐D‐glucoside synthase (IGS) that uses indoxyl and UDP‐glucose as its substrates. Indoxyl is possibly synthesized from indole by the catalysis of a monooxygenase, such as cytochrome P450 (CYP), whereas, sucrose synthase (SUS) or UDP‐glucose pyrophosphatase catalyzes the synthesis of UDP‐glucose. In the metabolism of indican, except IGS, the related proteins have not been identified to date; therefore, this study aimed to identify the specific proteins involved in the metabolism of the compound indican. Indoxyl is usually a very unstable substrate and is easily oxidized to indigo; due to its instability, it must be allowed to complete its indican synthesis reaction as fast as possible. In the plant secondary metabolism, the ER membrane‐associated CYPs that hydroxylate metabolites are well known. Furthermore, these CYPs may function as a scaffold for a temporary complex, known as metabolon, on the ER membrane. We assumed that the IGS also associates with some ER membrane proteins, because a part of IGS localizes on the microsome. We then performed protein–protein interaction analysis (coimmunoprecipitation and pull‐down assay) and detected some candidate proteins. Some proteins including CYPs and SUS were identified by the MS/MS analysis. We then examined the involvement of SUS in the metabolism of indican by performing protein–protein interaction analysis and cell fractionation using recombinant SUS.The full‐length cDNA of SUS gene obtained using the RACE‐PCR encodes a soluble cytosol protein composed of 806 amino acids. The recombinant SUS expressed in Escherichia coli was used to perform the experiments, as shown below. Firstly, we examined the intracellular localization of SUS using the cytosol and microsomal fractions prepared from the crude extract of leaf cells. Similar to IGS, SUS was also detected in both the fractions. The interaction between IGS and SUS was examined by coimmunoprecipitation using specific antibodies. We found that both proteins interacted with one another, suggesting that SUS might be a supply source for UDP‐glucose in the reaction involving the synthesis of indican. We are going to further discuss the data required to elucidate the entire indican metabolic pathway and to confirm the presence of its metabolon.