Secoisolariciresinol Dehydrogenase Purification, Cloning, and Functional Expression

Abstract

Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the ϳ32-kDa NAD-dependent secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (؊)-secoisolariciresinol into (؊)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of ϳ32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (؊)-secoisolariciresinol into (؊)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, secoisolariciresinol and matairesinol, has been elucidated. Last, both secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called "mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers.

Mechanistic and Structural Studies of Apoform, Binary, and Ternary Complexes of the Arabidopsis Alkenal Double Bond Reductase At5g16970

“…7C). The distances between the C-4 atom of the nicotinamide ring and the ␤-carbon of the ␣,␤-unsaturated double bond of 4-HNE (15) in the two subunits are 3.8 and 4.0 Å, respectively, which are approximately the same distances as for the p-coumaryl aldehyde (6) complex. Moreover, as in the ternary complex with p-coumaryl aldehyde (6), the conserved Tyr-53 is now in a hydrophobic interaction with the corresponding aliphatic chain of the bound 4-HNE (15), thereby again facilitating orientation of the substrate within the specificity pocket (Fig.…”

“…As a result, the corresponding conformation of the individual side chain and NADP ϩ did not change between the two ternary complexes. As observed in the p-coumaryl aldehyde (6) ternary complex, Tyr-260 is also within hydrogen bonding distance to the aldehydic oxygen of 4-HNE (15) and to the 2Ј-hydroxyl group of the nicotinamide ribose (Fig. 7C).…”

“…Examples of these include pinoresinol/lariciresinol reductases (13,14), secoisolariciresinol dehydrogenase (15)(16)(17), phenylcoumaran benzylic ether reductase (14,18), isoflavone reductase (14), cinnamyl alcohol dehydrogenases (19,20), and chavicol/p-anol and eugenol/isoeugenol synthases (21,22), as well as dirigent proteins (in the presence of auxiliary oxidative capacity) (23)(24)(25). These studies are part of broader goals aimed toward (i) systematically engineering selected enzyme substrate binding pockets in terms of potentially modifying them to be more specific for a particular metabolite/metabolic pathway and (ii) better understanding how these pathways in plants have evolved.…”

Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

“…The lignans are typically monolignol-derived dimers and are often found as either 8 -5Ј, 8 -8Ј, or 8-O-4Ј linked moieties that, depending upon the species, may be either optically active or racemic (1). It is only recently that the biosynthetic pathways to the lignans (3)(4)(5) and isoflavonoids (2) have been investigated, which in so doing resulted in the isolation and characterization of various lignan reductases, namely pinoresinol-lariciresinol reductases (PLRs) 1 (6,7) and phenylcoumaran benzylic ether reductases (PCBERs) (4), as well as isoflavone reductases (IFRs) (8,9) and their homologs.…”

“…For example, PLRs, which catalyze reduction of the 8 -8Ј linked lignan pinoresinol (Scheme 1, structure 1) (6,10), generate the precursors of cancer-preventative natural products, such as secoisolariciresinol diglucoside (structure 2) derivatives in flaxseed (Linum usitatissimum) (11,12) and matairesinol (structure 3)/secoisolariciresinol (structure 4) in a wide variety of plant species (5,11,13). Such lignans are considered to be metabolized in mammals into the cancer-preventing "mammalian" lignans, enterodiol (structure 5) and enterolactone (structure 6), with the latter metabolites being generated in the gut by the action of gastrointestinal flora such as Clostridia sp (14,15).…”