Evolution of Plant Defense Mechanisms

Abstract

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoidderived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent K m and V max values and observed kinetic 3 H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

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

“…Yet not only do they share high gene sequence similarity, but as described above they share similar physiological roles with their products (i.e. in plant defense) as well as apparently having comparable catalytic modes of action (4). However, the PLRs (6, 7) and IFRs (9) known so far catalyze enantiospecific conversions, whereas PCBER (4) only shows regiospecific discrimination, reducing both (ϩ)-and (Ϫ)-enantiomeric forms.…”

“…1C) (4). Although in this case such conversions proceed at relatively low enzymatic rates, both the (ϩ)-and (Ϫ)-enantiomeric forms of the substrates are utilized, and the 4-pro-R hydride is abstracted from the NADPH cofactor, thereby also classifying PCBER as a class A reductase (4).…”

“…dehydrodiconiferyl alcohol (structure 12) and dihydrodehydrodiconiferyl alcohol (structure 18) into isodihydrodehydrodiconiferyl alcohol (structure 19) and tetrahydrodehydrodiconiferyl alcohol (structure 20), respectively ( Fig. 1C) (4). Although in this case such conversions proceed at relatively low enzymatic rates, both the (ϩ)-and (Ϫ)-enantiomeric forms of the substrates are utilized, and the 4-pro-R hydride is abstracted from the NADPH cofactor, thereby also classifying PCBER as a class A reductase (4).…”

“…with dehydrodiconiferyl alcohol (structure 12) (4). Additionally, the isoflavone reductases are involved in the biosynthesis of various phytoalexins, such as (Ϫ)-medicarpin (structure 13) in alfalfa (Medicago sativa) (8), as well as in generating a number of heartwood-protective substances, e.g.…”

“…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.…”