Molecular cloning and functional identification of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Tripterygium wilfordii

Abstract

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the last step key enzyme of the methylerythritol phosphate (MEP) pathway, synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which is important for regulation of isoprenoid biosynthesis. Here the full-length cDNA of HDR, designated TwHDR (GenBank Accession No. KJ933412.1), was isolated from Tripterygium wilfordii for the first time. TwHDR has an open reading frame (ORF) of 1386 bp encoding 461 amino acids. TwHDR exhibits high homology with HDRs of other plants, with an N-terminal conserved domain and three conserved cysteine residues. TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant. Since loss-of-function E.coli hdr mutant is lethal, the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant. This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme. The expression of TwHDR was induced by methyl-jasmonate (MJ) in T. wilfordii suspension cells. The expression of TwHDR reached the highest level after 1 h of MJ treatment. These results indicate that we have identified a functional TwHDR enzyme, which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T. wilfordii.

Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, TwTPS27a and TwTPS27b, and Interaction Analysis with the Transcription Factor TwTGA1 from Tripterygium wilfordii

“…In recent years, the biosynthetic pathway of triptolide has been widely studied. Many biosynthetic enzyme genes have been isolated and functionally characterized, including not only the upstream pathway genes, such as TwHMGS [ 9 ], TwDXS1/2 [ 10 , 11 ], TwDXR [ 10 , 12 ], TwHDR [ 13 ], TwIDI [ 14 , 15 ], and TwGGPPS1/4/8 [ 16 , 17 ], but also the downstream pathway genes, such as TwTPS7/7v2/9/9v2 [ 18 , 19 ], TwTPS27/27v2 [ 18 , 19 ], and TwCYP728B70 [ 20 ]. However, less is known about the regulatory mechanism of key genes in triptolide biosynthesis in T. wilfordii , as compared with the progress of triptolide biosynthetic enzyme genes.…”

Triptolide: pharmacological spectrum, biosynthesis, chemical synthesis and derivatives

“…The TwHDR gene encodes the final enzyme of the MEP pathway, which is very important for regulating isoprene biosynthesis. The function of TwHDR was verified by a complementation assay in a mutant strain of E. coli with a defective HDR gene, and the expression of TwHDR in the cells in suspension was analyzed, and the results contributed to further analysis of the triptolide biosynthesis pathway 99 .…”

Ganglioside GD3 synthase (GD3S), a novel cancer drug target

“…The only reported GD3S inhibitor is triptolide. Triptolide, an epoxy diterpene lactone, is one of the main active components extracted from the roots, leaves, flowers and fruits of the Tripterygium 92 . It is a natural product with a variety of biological activities.…”