Pterocarpan synthase (PTS) structures suggest a common quinone methide–stabilizing function in dirigent proteins and proteins with dirigent-like domains

The biochemical activities of dirigent proteins (DPs) give rise to distinct complex classes of plant phenolics. DPs apparently began to emerge during the aquatic-to-land transition, with phylogenetic analyses revealing the presence of numerous DP subfamilies in the plant kingdom. The vast majority (>95%) of DPs in these large multigene families still await discovery of their biochemical functions. Here, we elucidated the 3D structures of two pterocarpan-forming proteins with dirigent-like domains. Both proteins stereospecifically convert distinct diastereomeric chiral isoflavonoid precursors to the chiral pterocarpans, (–)- and (+)-medicarpin, respectively. Their 3D structures enabled comparisons with stereoselective lignan– and aromatic terpenoid–forming DP orthologs. Each protein provides entry into diverse plant natural products classes, and our experiments suggest a common biochemical mechanism in binding and stabilizing distinct plant phenol–derived mono- and bis-quinone methide intermediates during different C–C and C–O bond–forming processes. These observations provide key insights into both their appearance and functional diversification of DPs during land plant evolution/adaptation. The proposed biochemical mechanisms based on our findings provide important clues to how additional physiological roles for DPs and proteins harboring dirigent-like domains can now be rationally and systematically identified. The biochemical activities of dirigent proteins (DPs) give rise to distinct complex classes of plant phenolics. DPs apparently began to emerge during the aquatic-to-land transition, with phylogenetic analyses revealing the presence of numerous DP subfamilies in the plant kingdom. The vast majority (>95%) of DPs in these large multigene families still await discovery of their biochemical functions. Here, we elucidated the 3D structures of two pterocarpan-forming proteins with dirigent-like domains. Both proteins stereospecifically convert distinct diastereomeric chiral isoflavonoid precursors to the chiral pterocarpans, (–)- and (+)-medicarpin, respectively. Their 3D structures enabled comparisons with stereoselective lignan– and aromatic terpenoid–forming DP orthologs. Each protein provides entry into diverse plant natural products classes, and our experiments suggest a common biochemical mechanism in binding and stabilizing distinct plant phenol–derived mono- and bis-quinone methide intermediates during different C–C and C–O bond–forming processes. These observations provide key insights into both their appearance and functional diversification of DPs during land plant evolution/adaptation. The proposed biochemical mechanisms based on our findings provide important clues to how additional physiological roles for DPs and proteins harboring dirigent-like domains can now be rationally and systematically identified. Dirigent protein (DP) (Latin: dirigere, to guide or align) (1Davin L.B. Wang H.-B. Crowell A.L. Bedgar D.L. Martin D.M. Sarkanen S. Lewis N.G. Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center.Science. 1997; 275 (8994027): 362-36710.1126/science.275.5298.362Crossref PubMed Scopus (515) Google Scholar) biochemical functions give entry into distinct complex plant phenol metabolic classes. DPs apparently began to functionally emerge during evolutionary transition of "primitive" aquatic plants to land. Phylogenetic analyses have indicated the presence of numerous subfamilies (i.e. DIR-a to DIR-h (2Ralph S.G. Jancsik S. Bohlmann J. Dirigent proteins in conifer defense II: extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.).Phytochemistry. 2007; 68 (17590394): 1975-199110.1016/j.phytochem.2007.04.042Crossref PubMed Scopus (85) Google Scholar, 3Corbin C. Drouet S. Markulin L. Auguin D. Lainé É. Davin L.B. Cort J.R. Lewis N.G. Hano C. A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.Plant Mol. Biol. 2018; 97 (29713868): 73-10110.1007/s11103-018-0725-xCrossref PubMed Scopus (30) Google Scholar)) thus far (Fig. 1) throughout the plant kingdom. DP multigene families currently span liverworts (e.g. Marchantia polymorpha) (4Bowman J.L. Kohchi T. Yamato K.T. Jenkins J. Shu S. Ishizaki K. Yamaoka S. Nishihama R. Nakamura Y. Berger F. Adam C. Aki S.S. Althoff F. Araki T. Arteaga-Vazquez M.A. et al.Insights into land plant evolution garnered from the Marchantia polymorpha genome.Cell. 2017; 171 (28985561): 287-30410.1016/j.cell.2017.09.030Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar), mosses (e.g. Physcomitrella patens (5Lang D. Ullrich K.K. Murat F. Fuchs J. Jenkins J. Haas F.B. Piednoel M. Gundlach H. Van Bel M. Meyberg R. Vives C. Morata J. Symeonidi A. Hiss M. Muchero W. et al.The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.Plant J. 2018; 93 (29237241): 515-53310.1111/tpj.13801Crossref PubMed Scopus (139) Google Scholar) and Sphagnum phallax (RRID:SCR_006507)), lycophytes (e.g. Selaginella moellendorffii (6Banks J.A. Nishiyama T. Hasebe M. Bowman J.L. Gribskov M. dePamphilis C. Albert V.A. Aono N. Aoyama T. Ambrose B.A. Ashton N.W. Axtell M.J. Barker E. Barker M.S. Bennetzen J.L. et al.The Selaginella genome identifies genetic changes associated with the evolution of vascular plants.Science. 2011; 332 (21551031): 960-96310.1126/science.1203810Crossref PubMed Scopus (561) Google Scholar)), gymnosperms (e.g. Picea sp., (2Ralph S.G. Jancsik S. Bohlmann J. Dirigent proteins in conifer defense II: extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.).Phytochemistry. 2007; 68 (17590394): 1975-199110.1016/j.phytochem.2007.04.042Crossref PubMed Scopus (85) Google Scholar) and Thuja plicata (7Kim M.K. Jeon J.-H. Davin L.B. Lewis N.G. Monolignol radical-radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications.Phytochemistry. 2002; 61 (12359517): 311-32210.1016/S0031-9422(02)00261-3Crossref PubMed Scopus (33) Google Scholar)), and angiosperms (e.g. Arabidopsis thaliana (2Ralph S.G. Jancsik S. Bohlmann J. Dirigent proteins in conifer defense II: extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.).Phytochemistry. 2007; 68 (17590394): 1975-199110.1016/j.phytochem.2007.04.042Crossref PubMed Scopus (85) Google Scholar, 7Kim M.K. Jeon J.-H. Davin L.B. Lewis N.G. Monolignol radical-radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications.Phytochemistry. 2002; 61 (12359517): 311-32210.1016/S0031-9422(02)00261-3Crossref PubMed Scopus (33) Google Scholar, 8Vassão D.G. Kim K.-W. Davin L.B. Lewis N.G. Lignans (neolignans) and allyl/propenyl phenols: biogenesis, structural biology, and biological/human health considerations.in: Mander L. Liu H.-W. Comprehensive Natural Products II Chemistry and Biology. Elsevier, Oxford, UK2010: 815-928Crossref Google Scholar, 9Kim K.-W. Moinuddin S.G.A. Atwell K.M. Costa M.A. Davin L.B. Lewis N.G. Opposite stereoselectivities of dirigent proteins in ArabidopsisSchizandra species.J. Biol. Chem. 2012; 287 (22854967): 33957-3397210.1074/jbc.M112.387423Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and Linum usitatissimum) (3Corbin C. Drouet S. Markulin L. Auguin D. Lainé É. Davin L.B. Cort J.R. Lewis N.G. Hano C. A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.Plant Mol. Biol. 2018; 97 (29713868): 73-10110.1007/s11103-018-0725-xCrossref PubMed Scopus (30) Google Scholar, 10Dalisay D.S. Kim K.-W. Lee C. Yang H. Rübel O. Bowen B.P. Davin L.B. Lewis N.G. Dirigent protein-mediated lignan and cyanogenic glucoside formation in flax seed: integrated omics and MALDI mass spectrometry imaging.J. Nat. Prod. 2015; 78 (25981198): 1231-124210.1021/acs.jnatprod.5b00023Crossref PubMed Scopus (63) Google Scholar) (Fig. 1); DPs are absent in algae and cyanobacteria (3Corbin C. Drouet S. Markulin L. Auguin D. Lainé É. Davin L.B. Cort J.R. Lewis N.G. Hano C. A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.Plant Mol. Biol. 2018; 97 (29713868): 73-10110.1007/s11103-018-0725-xCrossref PubMed Scopus (30) Google Scholar). However, most DPs (>95%) have no known biochemical function. All DPs and proteins harboring dirigent-like domains can be conveniently classified according to whether they contain the Pfam PF03018 domain (3Corbin C. Drouet S. Markulin L. Auguin D. Lainé É. Davin L.B. Cort J.R. Lewis N.G. Hano C. A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.Plant Mol. Biol. 2018; 97 (29713868): 73-10110.1007/s11103-018-0725-xCrossref PubMed Scopus (30) Google Scholar, 11El-Gebali S. Mistry J. Bateman A. Eddy S.R. Luciani A. Potter S.C. Qureshi M. Richardson L.J. Salazar G.A. Smart A. Sonnhammer E.L.L. Hirsh L. Paladin L. Piovesan D. Tosatto S.C.E. et al.The Pfam protein families database in 2019.Nucleic Acids Res. 2019; 47 (30357350): D427-D43210.1093/nar/gky995Crossref PubMed Scopus (1605) Google Scholar). To date, all DP subfamilies with known biochemical roles have been demonstrated to utilize different plant phenol substrates to gain entry into distinct plant phenol skeletal metabolic classes. The first DPs reported were the (+)- and (–)-pinoresinol–forming DPs affording entry into the lignan metabolic pathways (i.e. provided that one-electron (1e–) oxidation capacity was also present) (1Davin L.B. Wang H.-B. Crowell A.L. Bedgar D.L. Martin D.M. Sarkanen S. Lewis N.G. Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center.Science. 1997; 275 (8994027): 362-36710.1126/science.275.5298.362Crossref PubMed Scopus (515) Google Scholar, 7Kim M.K. Jeon J.-H. Davin L.B. Lewis N.G. Monolignol radical-radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications.Phytochemistry. 2002; 61 (12359517): 311-32210.1016/S0031-9422(02)00261-3Crossref PubMed Scopus (33) Google Scholar, 8Vassão D.G. Kim K.-W. Davin L.B. Lewis N.G. Lignans (neolignans) and allyl/propenyl phenols: biogenesis, structural biology, and biological/human health considerations.in: Mander L. Liu H.-W. Comprehensive Natural Products II Chemistry and Biology. Elsevier, Oxford, UK2010: 815-928Crossref Google Scholar, 9Kim K.-W. Moinuddin S.G.A. Atwell K.M. Costa M.A. Davin L.B. Lewis N.G. Opposite stereoselectivities of dirigent proteins in ArabidopsisSchizandra species.J. Biol. Chem. 2012; 287 (22854967): 33957-3397210.1074/jbc.M112.387423Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 10Dalisay D.S. Kim K.-W. Lee C. Yang H. Rübel O. Bowen B.P. Davin L.B. Lewis N.G. Dirigent protein-mediated lignan and cyanogenic glucoside formation in flax seed: integrated omics and MALDI mass spectrometry imaging.J. Nat. Prod. 2015; 78 (25981198): 1231-124210.1021/acs.jnatprod.5b00023Crossref PubMed Scopus (63) Google Scholar, 12Kim K.-W. Smith C.A. Daily M.D. Cort J.R. Davin L.B. Lewis N.G. Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.J. Biol. Chem. 2015; 290 (25411250): 1308-131810.1074/jbc.M114.611780Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 13Pickel B. Constantin M.-A. Pfannstiel J. Conrad J. Beifuss U. Schaller A. An enantiocomplementary dirigent protein for the enantioselective laccase-catalyzed oxidative coupling of phenols.Angew. Chem. Int. Ed. Engl. 2010; 49 (19946920): 202-20410.1002/anie.200904622Crossref PubMed Scopus (108) Google Scholar, 14Seneviratne H.K. Dalisay D.S. Kim K.-W. Moinuddin S.G.A. Yang H. Hartshorn C.M. Davin L.B. Lewis N.G. Non-host disease resistance response in pea (Pisum sativum) pods: biochemical function of DRR206 and phytoalexin pathway localization.Phytochemistry. 2015; 113 (25457488): 140-14810.1016/j.phytochem.2014.10.013Crossref PubMed Scopus (35) Google Scholar, 15Xia Z.-Q. Costa M.A. Proctor J. Davin L.B. Lewis N.G. Dirigent-mediated podophyllotoxin biosynthesis in Linum flavumPodophyllum peltatum.Phytochemistry. 2000; 55 (11130663): 537-54910.1016/S0031-9422(00)00242-9Crossref PubMed Scopus (82) Google Scholar) (Fig. 2A). In this way, the (+)- and (–)-pinoresinol-forming DPs (DIR-a subfamily members, Fig. 1) engender distinct stereoselective intermolecular couplings, in the presence of a 1e– oxidase or oxidant, of the prochiral coniferyl alcohol quinone methide (QM) free radicals so formed (i.e. to give the two distinct enantiomeric forms of pinoresinol, depending upon the Dir-a subfamily DP type). Conversely, in the absence of the DPs, only nonregiospecific and nonstereoselective phenoxy radical coupling occurs to afford a mixture of racemic products. (+)-Pinoresinol– or (–)-pinoresinol–forming DPs initially afford formation of enantiomeric bis-QM intermediates, via either si-si or re-re coupling, depending upon the DP in a particular plant species (see Fig. 2A). Following this C–C bond formation, these bis-QM intermediates can then undergo intramolecular cyclization (C–O bond formation) to give the lignans (+)- or (–)-pinoresinols, respectively (Fig. 2A). The (+)- and (–)-pinoresinol–forming DPs in subfamily DIR-a have been reported in a variety of plant systems, such as Forsythia intermedia (1Davin L.B. Wang H.-B. Crowell A.L. Bedgar D.L. Martin D.M. Sarkanen S. Lewis N.G. Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center.Science. 1997; 275 (8994027): 362-36710.1126/science.275.5298.362Crossref PubMed Scopus (515) Google Scholar), Podophyllum peltatum (15Xia Z.-Q. Costa M.A. Proctor J. Davin L.B. Lewis N.G. Dirigent-mediated podophyllotoxin biosynthesis in Linum flavumPodophyllum peltatum.Phytochemistry. 2000; 55 (11130663): 537-54910.1016/S0031-9422(00)00242-9Crossref PubMed Scopus (82) Google Scholar), western red cedar (T. plicata) (7Kim M.K. Jeon J.-H. Davin L.B. Lewis N.G. Monolignol radical-radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications.Phytochemistry. 2002; 61 (12359517): 311-32210.1016/S0031-9422(02)00261-3Crossref PubMed Scopus (33) Google Scholar), A. thaliana (8Vassão D.G. Kim K.-W. Davin L.B. Lewis N.G. Lignans (neolignans) and allyl/propenyl phenols: biogenesis, structural biology, and biological/human health considerations.in: Mander L. Liu H.-W. Comprehensive Natural Products II Chemistry and Biology. Elsevier, Oxford, UK2010: 815-928Crossref Google Scholar, 9Kim K.-W. Moinuddin S.G.A. Atwell K.M. Costa M.A. Davin L.B. Lewis N.G. Opposite stereoselectivities of dirigent proteins in ArabidopsisSchizandra species.J. Biol. Chem. 2012; 287 (22854967): 33957-3397210.1074/jbc.M112.387423Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Pickel B. Constantin M.-A. Pfannstiel J. Conrad J. Beifuss U. Schaller A. An enantiocomplementary dirigent protein for the enantioselective laccase-catalyzed oxidative coupling of phenols.Angew. Chem. Int. Ed. Engl. 2010; 49 (19946920): 202-20410.1002/anie.200904622Crossref PubMed Scopus (108) Google Scholar), flax (L. usitatissimum) (10Dalisay D.S. Kim K.-W. Lee C. Yang H. Rübel O. Bowen B.P. Davin L.B. Lewis N.G. Dirigent protein-mediated lignan and cyanogenic glucoside formation in flax seed: integrated omics and MALDI mass spectrometry imaging.J. Nat. Prod. 2015; 78 (25981198): 1231-124210.1021/acs.jnatprod.5b00023Crossref PubMed Scopus (63) Google Scholar), and pea (Pisum sativum) (12Kim K.-W. Smith C.A. Daily M.D. Cort J.R. Davin L.B. Lewis N.G. Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.J. Biol. Chem. 2015; 290 (25411250): 1308-131810.1074/jbc.M114.611780Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 14Seneviratne H.K. Dalisay D.S. Kim K.-W. Moinuddin S.G.A. Yang H. Hartshorn C.M. Davin L.B. Lewis N.G. Non-host disease resistance response in pea (Pisum sativum) pods: biochemical function of DRR206 and phytoalexin pathway localization.Phytochemistry. 2015; 113 (25457488): 140-14810.1016/j.phytochem.2014.10.013Crossref PubMed Scopus (35) Google Scholar). Pinoresinol is the biosynthetic entry point to many 8–8′-linked bioactive lignans, including several that have important roles in protecting against onset of different cancers and/or in clinically treating cancers (8Vassão D.G. Kim K.-W. Davin L.B. Lewis N.G. Lignans (neolignans) and allyl/propenyl phenols: biogenesis, structural biology, and biological/human health considerations.in: Mander L. Liu H.-W. Comprehensive Natural Products II Chemistry and Biology. Elsevier, Oxford, UK2010: 815-928Crossref Google Scholar). In a somewhat analogous manner, in aromatic terpenoid biosynthesis (16Liu J. Stipanovic R.D. Bell A.A. Puckhaber L.S. Magill C.W. Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein.Phytochemistry. 2008; 69 (18639908): 3038-304210.1016/j.phytochem.2008.06.007Crossref PubMed Scopus (45) Google Scholar), the (+)-gossypol–forming DP, GhDIR4 (17Effenberger I. Zhang B. Li L. Wang Q. Liu Y. Klaiber I. Pfannstiel J. Wang Q. Schaller A. Dirigent proteins from cotton (Gossypium sp.) for the atropselective synthesis of gossypol.Angew. Chem. Int. Ed. Engl. 2015; 54 (26460165): 14660-1466310.1002/anie.201507543Crossref PubMed Scopus (27) Google Scholar), in the DIR-b/d subfamily, helps engender stereoselective intermolecular coupling (C–C bond formation) of achiral hemigossypol moieties, provided there is an 1e– oxidase or oxidant. Again, in the absence of the DP, only racemic gossypol is formed. Stereoselective coupling, however, affords formation of the presumed chiral bis-QM, re-aromatization of which gives entry into the aromatic diterpenoid class, in this case (+)-gossypol (Fig. 2B). Gossypol occurs in leaves, roots, and seeds of cotton (Gossypium hirsutum) and imparts resistance against herbivorous insects and pathogens, but (–)-gossypol is toxic to animals. The ratio of (+)- to (–)-gossypol in cottons grown in the United Statesis ∼3:2, although it can be as high as 98:2 in moco cotton, such as in the variety marie-galante (16Liu J. Stipanovic R.D. Bell A.A. Puckhaber L.S. Magill C.W. Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein.Phytochemistry. 2008; 69 (18639908): 3038-304210.1016/j.phytochem.2008.06.007Crossref PubMed Scopus (45) Google Scholar). In pterocarpan (phytoalexin) biosynthesis studies, such as to (+)-pisatin in pea (Fig. 2C), it was deduced that DPs in the DIR-b/d subfamily were involved (18Celoy R.M. (+)-Pisatin Biosynthesis: From (–)-Enantiomeric Intermediates via an Achiral Isoflavene. University of Arizona, Tucson, AZ2013Google Scholar). 4H. Van Etten, personal communication. Based on this deduction, Dr. Tomoyoshi Akashi, following completion of his term as a visiting scientist in the research group of the late Hans Van Etten, examined formation of the structurally related (–)-medicarpin in licorice (Glycyrrhiza echinata) on returning to Japan. This led to the report of a medicarpin-forming DP (GePTS1) in the DIR-b/d family able to convert (3R,4R)-7,2′-dihydroxy-4′-methoxyisoflavanol (DMI) and (3S,4R)-DMI into (–)- and (+)-medicarpins, respectively, via lost of water and intramolecular C–O bond formation (19Uchida K. Akashi T. Aoki T. The missing link in leguminous pterocarpan biosynthesis is a dirigent domain-containing protein with isoflavanol dehydratase activity.Plant Cell Physiol. 2017; 58 (28394400): 398-40810.1093/pcp/pcw213Crossref PubMed Scopus (22) Google Scholar) (Fig. 2C). From its amino acid sequence, GePTS1 is a protein harboring dirigent-like domains. In addition to the DPs in the above diverse metabolic pathways, cell wall structural reinforcement via lignin deposition has been implicated to involve DIR-e subfamily members (e.g. Arabidopsis AtDIR10; Fig. 1) (20Hosmani P.S. Kamiya T. Danku J. Naseer S. Geldner N. Guerinot M.L. Salt D.E. Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.Proc. Natl. Acad. Sci. U. S. A. 2013; 110 (23940370): 14498-1450310.1073/pnas.1308412110Crossref PubMed Scopus (137) Google Scholar, 21Kamiya T. Borghi M. Wang P. Danku J.M.C. Kalmbach L. Hosmani P.S. Naseer S. Fujiwara T. Geldner N. Salt D.E. The MYB36 transcription factor orchestrates Casparian strip formation.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (26124109): 10533-1053810.1073/pnas.1507691112Crossref PubMed Scopus (111) Google Scholar) in the angiosperms at least. The latter DPs are reportedly part of supramolecular complexes in enabling another metabolic product, lignin, to be formed in Casparian band tissues. However, the actual physiological substrates that these DPs utilize have neither been identified nor demonstrated in vitro. The genes encoding DPs for entry points in pterocarpan, lignan, lignin biopolymer, and aromatic terpenoid biosynthesis are all of similar size. Of these DPs, the DIR-e lignin-forming DPs have much longer β1-β2 loops in their 3D structures, when compared with other DP's (e.g. DRR206 (12Kim K.-W. Smith C.A. Daily M.D. Cort J.R. Davin L.B. Lewis N.G. Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.J. Biol. Chem. 2015; 290 (25411250): 1308-131810.1074/jbc.M114.611780Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), AtDIR6 (22Gasper R. Effenberger I. Kolesinski P. Terlecka B. Hofmann E. Schaller A. Dirigent protein mode of action revealed by the crystal structure of AtDIR6.Plant Physiol. 2016; 172 (27756822): 2165-217510.1104/pp.16.01281Crossref PubMed Scopus (24) Google Scholar), and GhDIR4 (17Effenberger I. Zhang B. Li L. Wang Q. Liu Y. Klaiber I. Pfannstiel J. Wang Q. Schaller A. Dirigent proteins from cotton (Gossypium sp.) for the atropselective synthesis of gossypol.Angew. Chem. Int. Ed. Engl. 2015; 54 (26460165): 14660-1466310.1002/anie.201507543Crossref PubMed Scopus (27) Google Scholar). However, the biochemical significance of these much longer β1-β2 loops is currently unknown. Many DP sequences contain canonical N-linked glycosylation motifs, and some have been confirmed experimentally as being post-translationally glycosylated, such as FiDIR1 (23Gang D.R. Costa M.A. Fujita M. Dinkova-Kostova A.T. Wang H.-B. Burlat V. Martin W. Sarkanen S. Davin L.B. Lewis N.G. Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis.Chem. Biol. 1999; 6 (10074466): 143-15110.1016/S1074-5521(99)89006-1Abstract Full Text PDF PubMed Scopus (162) Google Scholar), DRR206 (12Kim K.-W. Smith C.A. Daily M.D. Cort J.R. Davin L.B. Lewis N.G. Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.J. Biol. Chem. 2015; 290 (25411250): 1308-131810.1074/jbc.M114.611780Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 14Seneviratne H.K. Dalisay D.S. Kim K.-W. Moinuddin S.G.A. Yang H. Hartshorn C.M. Davin L.B. Lewis N.G. Non-host disease resistance response in pea (Pisum sativum) pods: biochemical function of DRR206 and phytoalexin pathway localization.Phytochemistry. 2015; 113 (25457488): 140-14810.1016/j.phytochem.2014.10.013Crossref PubMed Scopus (35) Google Scholar), AtDIR6 (9Kim K.-W. Moinuddin S.G.A. Atwell K.M. Costa M.A. Davin L.B. Lewis N.G. Opposite stereoselectivities of dirigent proteins in ArabidopsisSchizandra species.J. Biol. Chem. 2012; 287 (22854967): 33957-3397210.1074/jbc.M112.387423Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Gasper R. Effenberger I. Kolesinski P. Terlecka B. Hofmann E. Schaller A. Dirigent protein mode of action revealed by the crystal structure of AtDIR6.Plant Physiol. 2016; 172 (27756822): 2165-217510.1104/pp.16.01281Crossref PubMed Scopus (24) Google Scholar), and GhDIR4 (17Effenberger I. Zhang B. Li L. Wang Q. Liu Y. Klaiber I. Pfannstiel J. Wang Q. Schaller A. Dirigent proteins from cotton (Gossypium sp.) for the atropselective synthesis of gossypol.Angew. Chem. Int. Ed. Engl. 2015; 54 (26460165): 14660-1466310.1002/anie.201507543Crossref PubMed Scopus (27) Google Scholar). On the other hand, the medicarpin-forming DP appears to have no requirement for post-translational glycosylation. With the availability of structures of stereoselective medicarpin-forming DPs (nonglycosylated), stereoselective lignan-forming DPs (both apparently requiring post-translational glycosylation for stability), and a homology-modeled aromatic diterpenoid DP (GhDIR4), it was instructive to probe and compare the mechanistic biochemical features of these distinct DP types. Described herein are the 3D structures of two stereoselective pterocarpan-forming DPs from pea and licorice, which preferentially produce either (+)- or (–)-medicarpin, depending on the substrate (Fig. 2C). These findings are discussed in the context of this DP type, which has dirigent-like (amino acid sequence similarity) domains as compared with the stereoselective lignan and aromatic terpenoid-forming DPs. Of particular interest was whether there was a common DP biochemical mechanism and, if so, what were the underlying mechanistic principles involved. We describe that pterocarpan synthases, containing dirigent-like domains, initially engender mono-QM formation from their chiral substrates, this being followed by intramolecular cyclization (C–O bond formation) to afford entry into the pterocarpan natural product (phytoalexin) class. These differ from the other dirigent protein types, which instead initially enable stereoselective, one-electron, intermolecular coupling (C–C bond formation) of two identical achiral aromatic precursors to give chiral bis-QMs. The latter then either undergo intramolecular cyclization (C–O bond formation) or re-aromatization, respectively, to generate lignan and aromatic diterpenoid natural product classes. The P. sativum "Cam_eor" Unigene set (24Alves-Carvalho S. Aubert G. Carrère S. Cruaud C. Brochot A.-L. Jacquin F. Klein A. Martin C. Boucherot K. Kreplak J. da Silva C. Moreau S. Gamas P. Wincker P. Gouzy J. et al.Full-length de novo assembly of RNA-seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species.Plant J. 2015; 84 (26296678): 1-1910.1111/tpj.12967Crossref PubMed Scopus (93) Google Scholar) was searched using GePTS1 as query, which resulted in a gene (PsCam039127) being selected as possibly encoding a medicarpin-forming DP. Trivially named PsPTS1, it has ∼92%/85% sequence similarity/identity to GePTS1 at the amino acid level. Fig. 3 shows amino acid sequence alignments of the medicarpin-forming DPs (GePTS1 and PsPTS1), the (+)- and (–)-pinoresinol–forming DPs (DRR206 and AtDIR6) from P. sativum and A. thaliana, respectively, and the aromatic diterpenoid (+)-gossypol–forming DP (GhDIR4) in G. hirsutum. GePTS1 and PsPTS1 coding sequences were individually codon-optimized for Escherichia coli, with each synthetic gene cloned into the pET101/D-TOPO® E. coli expression vector harboring a C-terminal 6× polyhistidine region. The vector constructs were then each used to transform E. coli BL21 (DE3) cells. After induction with isopropyl 1-thio-β-d-galactopyranoside, the resulting recombinant His-tagged proteins were individually purified to apparent homogeneity (Fig. S1A) by use of metal-chelating affinity chromatography. Gel-permeation chromatography (GPC) was next carried out on a TSKgel G3000SWXL column, precalibrated with molecular weight standards, to determine the oligomeric state of both PTSs. GePTS1 and PsPTS1, in solution, exist mainly as trimers (∼68.0 kDa), with (because of association/aggregation) a small amount of higher-molecular weight entities also being evident (roughly corresponding to 410–500 kDa). Next, both GePTS1 and PsPTS1 DPs were used in assays with racemic mixtures of the diastereomers obtained through chemical synthesis from racemic vestitone (see "Experimental procedures") (i.e. either cis-DMI ((3R,4R) and (3S,4S)) or trans-DMI ((3S,4R) and (3R,4S)), respectively), with substrates and products easily resolved by chiral column chromatography (Chiral OJ column, Chiral Technologies). As reported previously (19Uchida K. Akashi T. Aoki T. The missing link in leguminous pterocarpan biosynthesis is a dirigent domain-containing protein with isoflavanol dehydratase activity.Plant Cell Physiol. 2017; 58 (28394400): 398-40810.1093/pcp/pcw213Crossref PubMed Scopus (22) Google Scholar), GePTS1 converted either (3R,4R)-DMI or (3S,4R)-DMI into (–)- or (+)-medicarpin, respectively (Fig. S2, B and I). The pea medicarpin-forming DP (PsPTS1) catalyzed the same conversions (Fig. S2, C and J). Control assays (no DP present) gave smaller amounts of racemic medicarpin products (Fig. S2, A and H) because of nonenzymatic conversion of cis- and trans-DMI. Kinetic data for both DPs were next obtained as follows: assays were carried out in triplicate at 10 concentrations of cis-DMI ((3R,4R) and (3S,4S)) and trans-DMI ((3S,4R) and (3R,4S)) for 5 min. Triplicate assays were also carried out in the absence of DPs to account for the nonenzymatic conversion of cis- and trans-DMI. From these determinations, GePTS1 preferentially utilized the cis-DMI (3R,4R) isomer, whereas the (3S,4S) cis-DMI was not con