Pinoresinol–lariciresinol reductases with different stereospecificity from Linum album and Linum usitatissimum

Abstract

Recently it was found that cell cultures and plants of Linum species contain lignans of various chemical structures. The stereochemistry of these compounds differ among species. Cell cultures of L. album accumulate (−)-podophyllotoxin together with pure (−)-secoisolariciresinol. The presence of both enantiomers of the precursor pinoresinol indicates that in L. album cell cultures the reactions from pinoresinol to secoisolariciresinol are the first steps determining enantiospecificity in biosynthesis of podophyllotoxin. Seeds of L. usitatissimum contain almost enantiomerically pure (+)-secoisolariciresinoldiglucosid derived from (+)-secoisolariciresinol. A cell culture of this species contains a mixture of both enantiomers of pinoresinol and pure (+)-secoisolariciresinol. In order to get more insight into the mechanism of (−)- and (+)-secoisolariciresinol biosynthesis, respectively, we isolated a cDNA encoding pinoresinol–lariciresinol reductase (PLR) from L. album. The heterologously expressed PLR-La1 converts only (+)-pinoresinol into (−)-secoisolariciresinol. In contrast, the heterologously expressed PLR from L. usitatissimum converts only (−)-pinoresinol to (+)-secoisolariciresinol confirming the results from others. Comparison of all available PLR protein sequences resulted in a few amino acids which may be responsible for the action of the PLRs with respect to the different enantioselectivity. A mutagenesis approach could not confirm this hypothesis. Aspects about the evolution of PLRs are discussed.