(+)‐Pinoresinol/(−)‐lariciresinol reductase from Linum perenne Himmelszelt involved in the biosynthesis of justicidin B

Abstract

A cDNA encoding a pinoresinol-lariciresinol reductase PLR (PLR-Lp1) was isolated from a cell culture of Linum perenne Himmelszelt accumulating the arylnaphthalene lignan justicidin B. The recombinant PLR-Lp1 prefers (+)-pinoresinol in the first reaction step, but (À)-lariciresinol in the second step. Therefore, it is the first PLR described with opposite enantiospecificity within the two reaction steps catalysed by PLRs. Hairy root lines transformed with an ihpRNAi construct to suppress plr gene expression show less mRNA accumulation for the plr-Lp1 gene and PLR enzyme activity. Justicidin B accumulation was reduced down to 24% in comparison to control lines showing the involvement of PLR-Lp1 in the biosynthesis of justicidin B.

Characterization of Arabidopsis thaliana Pinoresinol Reductase, a New Type of Enzyme Involved in Lignan Biosynthesis

“…It was proposed that PLR and SIRD are key enzymes that control the enantiomeric compositions of the lignans in the pathway (8). However, the studies of gene expression levels to examine the working hypothesis have been very difficult, because gene transformation and regeneration systems for plants that are known as lignan producers were very limited, although very recently gene silencing systems for the hairy root lines of the lignan-producing Linum perenne have been established (20).…”

Bioconversion of Pinoresinol into Matairesinol by Use of Recombinant Escherichia coli

“…A segment of plr in P. pleianthum Hance was amplified by RT-PCR based on the conserved part of the plr sequences in Forsythia intermedia (U81158), Thuja plicata (AF242503 and AF242503), Linum album (AJ849358), and Linum usitatissimum (AJ849359), resulting in a 320-bp cDNA fragment, the deduced protein sequence of which showed high similarity to those of all of the PLRs described above and that determined later for Linum perenne (20). A 933-bp cDNA containing the fulllength coding sequence, plr-Pp (GenBank accession number KJ000045), was cloned by 5=-and 3=-RACE PCR.…”

“…The enzyme PLR has been identified in Forsythia intermedia (15), Thuja plicata (18), Linum perenne (20), L. album, and L. usitatissimum (19). We cloned plr cDNA from P. pleianthum Hance.…”

“…The enzyme activity assay for recombinant PLR (rPLR) was modified from that in a previous report (20). The assay mixture (250 l) containing 55 M, 110 M, 167 M, 223 M, and 279 M (ϩ)-pinoresinol or (ϩ)-lariciresinol, 2.5 mM NADPH, 10 g of rPLR, and 0.1 M potassium phosphate buffer, pH 7.1, was incubated at 30°C for 20 min.…”

“…A binary buffer system (solvent A water containing 0.01% phosphoric acid, solvent B containing acetonitrile) was used to create the mobile-phase gradient. The analytes were separated using 25% solvent B for the first 25 min and then linear gradients of 25 to 43% solvent B for 18 min, 43 to 55% for 3 min, 55 to 70% for 8 min, 70 to 25% for 2 min, and 25% for 4 min at a flow rate of 1 ml/min (20) with detection at 280 nm.…”