Abstract
Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (-)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.
Highlights
Dirigent protein (DP) discovery gave new paradigm for monolignol-derived coupling in planta
Determination of the PsDRR206 three-dimensional structure, and its analysis suggests that the corresponding trimer has three identical substrate binding sites that are spatially distributed so that intermolecular coupling apparently cannot occur between two distinct substrate binding sites in the trimer model
This trimer observation is in agreement with our earlier studies (3), where the Forsythia DP was considered as a ϳ78-kDa trimer based on gel filtration analyses, in contrast to subsequent mass spectrometric data obtained, which had suggested a dimeric protein (38)
Summary
Dirigent protein (DP) discovery gave new paradigm for monolignol-derived coupling in planta. One electron oxidation (e.g. catalyzed by laccases, peroxidases) is envisaged to generate the corresponding coniferyl alcohol (1)-derived free radical intermediate (CA1⁄7), which binds to the DPs. Bound substrate orientation subsequently occurs in such a way as to enable 8 – 8Ј coupling at the si-si face with concomitant intramolecular cyclization to afford (ϩ)-pinoresinol (Fig. 1A, 2a), such as in Forsythia intermedia (3), Thuja plicata (4), Schizandra chinensis (5), or pea (Pisum sativum) (7), or alternatively re-re coupling to give the (Ϫ)antipode (Fig. 1A, 2b) as in Arabidopsis (1, 5, 6) and flax (Linum usitatissimum).[3] Of these, the (ϩ)-pinoresinol-forming DP in pea is annotated as PsDRR206 (9), and the (Ϫ)-pinoresinolforming DP in Arabidopsis thaliana is named AtDIR6 (5).
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