| (+)-Pinoresinol/(+)-lariciresinol reductase catalyzes the first known example of highly unusual NADPH-dependent benzylic ether reductions in plants. It was discovered in Forsythia intermedia and is a pivotal branching point enzyme in the formation of plant lignans. Lignans exhibit important pharmacological roles, such as the phytoestrogens in high-fiber diets and various antitumor (e.g. podophyllotoxin) and antiviral (e.g. trachelogenin) agents, as well as crucial functions in plant defense. Entry into lignan enzymology has been gained by the $sim$3200 fold purification, to apparent electrophoretic homogeneity, of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase. Both isoforms have comparable molecular weights and kinetic (K$sb{rm m}$, V$sb{rm max}$) properties, with stereospecific hydride transfer being catalyzed where the incoming hydride takes up the proR position, thereby eliminating the possibility of random hydride delivery to a planar quinone methide intermediate. As might be expected, the mode of hydride abstraction from NADPH is also stereospecific: using (4R-$sp3$H) and (4S-$sp3$H) NADPH, it was found that only the 4 pro-R hydride was abstracted from the nicotinamide ring of the cofactor. The gene, encoding the reductase has been cloned and heterologously expressed in Escherichia coli as a $beta$-galactosidase fusion protein. The native protein has a calculated molecular mass of 34.9 kD and catalyzes the sequential, enantiospecific, NADPH-dependent conversion of (+)-pinoresinol to (-)-secoisolariciresionol via (+)-lariciresinol.;The amino acid sequence of (+)-pinoresinol/(+)-lariciresinol reductase reveals strong homology to isoflavone reductases, which catalyze important steps in the biosynthesis of phytoalexins, e.g., (-)-medicarpin. Since the latter are also products of phenylpropanoid-acetate metabolism and display plant defense and phytoestrogen properties, it is tempting to speculate that the isoflavone reductases may have evolved from (+)-pinoresinol/(+)-lariciresinol reductase. This is proposed because the lignans are widely distributed in the plant kingdom, including "primitive" plants, whereas the isoflavonoids appear to be mainly restricted to the Leguminosae. |
