CDNA cloning and characterization of UDP‐glucosyltransferase from Indigofera tinctoria

Abstract

Indigofera tinctoria is a cultivated plant that is utilized as a source of the natural blue dye, indigo. The plant produces a colorless secondary metabolite known as indican (indoxyl β‐D‐glucoside). Following its released from the cells, indican is enzymatically degraded into indoxyl and glucose. Indoxyl is an unstable compound that is easily oxidized and dimerizes to produce the stable indigo. Unlike the degradation reaction of indican, there is limited knowledge about indican biosynthesis pathway in the cells. We have previously reported that Polygonum tinctorium, an alternative indigo‐producing plant, has UDP‐glucosyltransferase (UGT) that catalyzes the synthesis reaction of indican from indoxyl and UDP‐glucose. However, there are several subjects that need to be studied including the cDNA cloning, the subcellular localization, the transport pathway of substrate and product during indican metabolism, etc. We hypothesize that the indican metabolic pathway in Indigofera is similar to that in Polygonum. Here, we report cDNA cloning and characterization of UGT from Indigofera tinctoria. The sample containing UGT that was partially purified from Indigofera leaves was separated on SDS‐PAGE gel. After staining the gel with CBB, several bands were cut off and analyzed by peptide mass fingerprinting. We identified two fragments homologous to UGT from the transcriptomic data that was generated from Indigofera leaf tissue. Based on the sequence of the fragments, RACE method was performed, leading to amplification of two types of full‐length Ugt cDNAs, itUgt1 and itUgt2. The polypeptide sequence of 477 amino acids encoded by itUgt1 shows high homology (max identity is 89%) with the 475 amino acids encoded by itUgt2. Their primary structures contain the motif of UDP‐glucoronosyl and ‐glucosyl transferase and show the high homology to some UGTs from other plants. To analyze the properties and functions of itUGT1 and itUGT2, the recombinant proteins were expressed in E. coli and purified. The antibody was raised against recombinant itUGT2. The recombinant itUGT1 and itUGT2 show high activity of indican synthesis as reported for the Polygonum enzyme. Although there was no difference between values of optimum pH, itUGT2 was more stable with temperature variations than itUGT1. Further, the expression of itUGT proteins and mRNA levels in various tissues from Indigofera were examined by Western blot and semi‐quantitative RT‐PCR, respectively. Consequently, it was observed that UGT1 synthesizes indican in Indigofera tissues and not UGT2. Further work on cell fractionation, for determining the subcellular localization of itUGTs, is in progress