Stereochemistry in lignan biosynthesis

Lignans show a wide variety of structural types and enantiomeric forms. Despite this large diversity, the first biosynthetic steps seem to be universally valid. Two molecules of coniferyl alcohol are dimerised to pinoresinol (Pino) which is further reduced via lariciresinol (Lari) to seco-isolariciresinol (Seco) by the pinoresinol-lariciresinol reductase (PLR). In cell cultures of Linum album and L. usitatissimum the enantiomeric purity is determined by the enantiospecificity of the PLR. In order to understand the evolutionary background for these differences in enantio-specificity we cloned PLRs and related enzymes like phenyl-coumaran-benzylic-ether reductases (PCBERs) from Linum species accumulating different types of lignans and of Arabidopsis thaliana. The functionality of the recombinant proteins was distinguished by using dehydrodiconiferyl alcohol (the substrate for PCBERs) and racemic Pino as substrates. The enantiospecificity of PLR was determined by chiral column chromatography of the remaining Pino and formed Lari and Seco from reactions with different protein concentrations or after different reaction times. We found a broad variation in enantiospecifity. The comparison between a phylogenetic tree of PLRs with plant molecular phylogeny leads to the assumption that only a few amino acids have to be exchanged to alter the enantiospecificity of PLRs. We figure out which amino acids in the protein are responsible for enantiospecificity by a mutagenesis approach. We use the recombinant PLR from L. album which strongly prefers (+)-Pino (R,R-configuration) as substrate to form preferentially (-)-Seco (R,R-configuration) in comparison to a PLR from L. usitatissimum which works with the opposite preference as model.

Acknowledgements: Financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

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