title: Opposite stereoselectivities of dirigent proteins in Arabidopsis and schizandra species.
abstract: How stereoselective monolignol-derived phenoxy radical-radical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (-)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (-)-pinoresinol-forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidase reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (-)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (-)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinol-forming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
authors.forename: Kye-Won
authors.initials: KW
authors.lastname: Kim
authors.forename: Syed G A
authors.initials: SG
authors.lastname: Moinuddin
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Atwell
authors.forename: Michael A
authors.initials: MA
authors.lastname: Costa
authors.forename: Laurence B
authors.initials: LB
authors.lastname: Davin
authors.forename: Norman G
authors.initials: NG
authors.lastname: Lewis
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D005663:Furans / Q000378:metabolism; D017705:Lignans / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032141:Schisandra / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D005663:Furans; D017705:Lignans; D010940:Plant Proteins; C103298:pinoresinol