(-PINORESINOL())(+)-LARICIRESINOL REDUCTASE FROM FORSYTHIA-INTERMEDIA- PROTEIN-PURIFICATION, CDNA CLONING, HETEROLOGOUS EXPRESSION AND COMPARISON TO ISOFLAVONE REDUCTASE/

Lignans are a widely distributed class of natural products, whose func tions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyl lotoxin and enterodiol, have important roles in cancer chemotherapy an d prevention, respectively. Entry into lignan enzymology has been gain ed by the similar to 3000-fold purification of two isoforms of (+)-pin oresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable (similar to 34.9 kDa) molecu lar mass and kinetic (V-max/K-m) properties and catalyze sequential, N ADPH-dependent, stereospecific, hydride transfers where the incoming h ydride takes up the pro-R position. The gene encoding (+)-pinoresinol/ (+)-lariciresinol reductase has been cloned and the recombinant protei n heterologously expressed as a functional beta-galactosidase fusion p rotein. Its amino acid sequence reveals a strong homology to isoflavon e reductase, a key branchpoint enzyme in isoflavonoid metabolism and p rimarily found in the Fabaceae (angiosperms). This is of great evoluti onary significance since both lignans and isoflavonoids have comparabl e plant defense properties, as well as similar roles as phytoestrogens . Given that lignans are widespread from primitive plants onwards, whe reas the isoflavone reductase-derived isoflavonoids are mainly restric ted to the Fabaceae, it is tempting to speculate that this branch of t he isoflavonoid pathway arose via evolutionary divergence from that gi ving the lignans.