title: Dirigent Protein Mode of Action Revealed by the Crystal Structure of AtDIR6.
abstract: Dirigent proteins impart stereoselectivity to phenoxy radical coupling reactions in plants and, thus, play an essential role in the biosynthesis of biologically active natural products. This includes the regioselective and enantioselective coupling and subsequent cyclization of two coniferyl alcohol radicals to pinoresinol as the committed step of lignan biosynthesis. The reaction is controlled by dirigent proteins, which, depending on the species and protein, direct the reaction to either (+)- or (-)-pinoresinol. We present the crystal structure of the (-)-pinoresinol forming DIRIGENT PROTEIN6 (AtDIR6) from Arabidopsis (Arabidopsis thaliana) with data to 1.4 Å resolution. The structure shows AtDIR6 as an eight-stranded antiparallel β-barrel that forms a trimer with spatially well-separated cavities for substrate binding. The binding cavities are two lobed, exhibiting two opposing pockets, each lined with a set of hydrophilic and potentially catalytic residues, including essential aspartic acids. These residues are conserved between (+) and (-)-pinoresinol-forming DIRs and required for activity. The structure supports a model in which two substrate radicals bind to each of the DIR monomers. With the aromatic rings fixed in the two pockets, the propionyl side chains face each other for radical-radical coupling, and stereoselectivity is determined by the exact positioning of the side chains. Extensive mutational analysis supports a previously unrecognized function for DIRs in catalyzing the cyclization of the bis-quinone methide reaction intermediate to yield (+)- or (-)-pinoresinol.
authors.affiliation: Ruhr University Bochum, AG Protein Crystallography, Biophysics, 44801 Bochum, Germany (R.G., B.T., E.H.).|University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70593 Stuttgart, Germany (I.E., A.S); and.|University of Wroclaw, Laboratory of Biophysics, Faculty of Biotechnology, 50-383 Wroclaw, Poland (P.K.).
authors.forename: Raphael
authors.initials: R
authors.lastname: Gasper
authors.affiliation: Ruhr University Bochum, AG Protein Crystallography, Biophysics, 44801 Bochum, Germany (R.G., B.T., E.H.).|University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70593 Stuttgart, Germany (I.E., A.S); and.|University of Wroclaw, Laboratory of Biophysics, Faculty of Biotechnology, 50-383 Wroclaw, Poland (P.K.).
authors.forename: Isabelle
authors.initials: I
authors.lastname: Effenberger
authors.affiliation: Ruhr University Bochum, AG Protein Crystallography, Biophysics, 44801 Bochum, Germany (R.G., B.T., E.H.).|University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70593 Stuttgart, Germany (I.E., A.S); and.|University of Wroclaw, Laboratory of Biophysics, Faculty of Biotechnology, 50-383 Wroclaw, Poland (P.K.).
authors.forename: Piotr
authors.identifier: 0000-0001-6034-8445
authors.initials: P
authors.lastname: Kolesinski
authors.affiliation: Ruhr University Bochum, AG Protein Crystallography, Biophysics, 44801 Bochum, Germany (R.G., B.T., E.H.).|University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70593 Stuttgart, Germany (I.E., A.S); and.|University of Wroclaw, Laboratory of Biophysics, Faculty of Biotechnology, 50-383 Wroclaw, Poland (P.K.).
authors.forename: Barbara
authors.initials: B
authors.lastname: Terlecka
authors.affiliation: Ruhr University Bochum, AG Protein Crystallography, Biophysics, 44801 Bochum, Germany (R.G., B.T., E.H.); eckhard.hofmann@bph.rub.de andreas.schaller@uni-hohenheim.de.|University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70593 Stuttgart, Germany (I.E., A.S); and eckhard.hofmann@bph.rub.de andreas.schaller@uni-hohenheim.de.|University of Wroclaw, Laboratory of Biophysics, Faculty of Biotechnology, 50-383 Wroclaw, Poland (P.K.) eckhard.hofmann@bph.rub.de andreas.schaller@uni-hohenheim.de.
authors.forename: Eckhard
authors.identifier: 0000-0003-4874-372X
authors.initials: E
authors.lastname: Hofmann
authors.affiliation: Ruhr University Bochum, AG Protein Crystallography, Biophysics, 44801 Bochum, Germany (R.G., B.T., E.H.); eckhard.hofmann@bph.rub.de andreas.schaller@uni-hohenheim.de.|University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70593 Stuttgart, Germany (I.E., A.S); and eckhard.hofmann@bph.rub.de andreas.schaller@uni-hohenheim.de.|University of Wroclaw, Laboratory of Biophysics, Faculty of Biotechnology, 50-383 Wroclaw, Poland (P.K.) eckhard.hofmann@bph.rub.de andreas.schaller@uni-hohenheim.de.
authors.forename: Andreas
authors.identifier: 0000-0001-6872-9576
authors.initials: A
authors.lastname: Schaller
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D003500:Cyclization; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D029681:Arabidopsis Proteins; C586363:DIR6 protein, Arabidopsis; D010636:Phenols; C010559:coniferyl alcohol