(+)-Pisatin Biosynthesis: From (-) Enantiomeric Intermediates via an Achiral Isoflavene

Persistent Link:
http://hdl.handle.net/10150/293423
Title:
(+)-Pisatin Biosynthesis: From (-) Enantiomeric Intermediates via an Achiral Isoflavene
Author:
Celoy, Rhodesia Mateo
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Pterocarpan phytoalexins are antimicrobial compounds produced by legumes when challenged by biotic stresses. Most legumes produce pterocarpan phytoalexins with (-)-stereochemistry but pea (Pisum sativum L.) produces as its major phytoalexin (+)-pisatin. Pea also occasionally produces a minor amount of (-)-maackiain as a pterocarpan phytoalexin, and studies on the biosyntheses of (+)-pisatin and (-)-maackiain have shown that up to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and 7,2'-dihydroxy-4',5'-methyl-enedioxyisoflavanol [(-)-DMDI]they have common intermediates with (-)-DMDI being where the two pathways diverge. The final step in (+)-pisatin biosynthesis is the methylation of (+)-6a-hydroxymaackiain [(+)-6a-HMK] by 6a-hydroxymaackiain methyltransferase (HMM2) but the steps from (-)-DMDI to (+)-6a-HMK are unknown.The shifting of the stereochemistry from (-)-DMDI to (+)-6a-HMK has been proposed to involve the achiral isoflavene, 7, 2'-dihydroxy-4', 5'-methylene-dioxyisoflav-3-ene (DMDIF). In this dissertation, I have shown that cis-(-)-DMDI is the enzymatic product of (-)-sophorol, and is the precursor of DMDIF which is produced by the dehydration activity of "isoflavene synthase" (IFVS). IFVS activity was not observed in elicited tissues of alfalfa, chickpea, beans, pepper, and broccoli, plants that do not produce (+) pterocarpans. Partial purification of IFVS demonstrated that it is either large in size or tightly complexed with other proteins. The SDS-PAGE of the 29-fold purified product revealed 12 major bands that aggregated into 3 bands in the non-denaturing PAGE. IFVS activity was in band 3 which co-migrated with marker proteins of>100 kDa in size. Proteins identified from LC-MS/MS peptide sequences of the proteins in band 3, when compared to three protein databases, did not identify any proteins with an enzymatic activity expected for IFVS. A disease resistance-response protein (a dirigent-like protein) and two protein-binding proteins were the most abundantly detected proteins in the pea transcriptome-translated database. Also, four of the known enzymes (isoflavone reductase, HMM1, HMM2, and sophorol reductase) involved in (+)-pisatin biosynthesis were among the proteins identified. It may be that IFVS is associated with these other proteins as a complex in vitro and in vivo. The lack of detection of IFVS in the databases could be because it has not yet been sequenced as it functions in a rare biosynthetic pathway.
Type:
text; Electronic Dissertation
Keywords:
Isoflavonoid; Pea; Phytoalexin; Pisatin; Pisum sativum L; Plant Pathology; Isoflavene
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Plant Pathology
Degree Grantor:
University of Arizona
Advisor:
VanEtten, Hans D.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.title(+)-Pisatin Biosynthesis: From (-) Enantiomeric Intermediates via an Achiral Isoflaveneen_US
dc.creatorCeloy, Rhodesia Mateoen_US
dc.contributor.authorCeloy, Rhodesia Mateoen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractPterocarpan phytoalexins are antimicrobial compounds produced by legumes when challenged by biotic stresses. Most legumes produce pterocarpan phytoalexins with (-)-stereochemistry but pea (Pisum sativum L.) produces as its major phytoalexin (+)-pisatin. Pea also occasionally produces a minor amount of (-)-maackiain as a pterocarpan phytoalexin, and studies on the biosyntheses of (+)-pisatin and (-)-maackiain have shown that up to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and 7,2'-dihydroxy-4',5'-methyl-enedioxyisoflavanol [(-)-DMDI]they have common intermediates with (-)-DMDI being where the two pathways diverge. The final step in (+)-pisatin biosynthesis is the methylation of (+)-6a-hydroxymaackiain [(+)-6a-HMK] by 6a-hydroxymaackiain methyltransferase (HMM2) but the steps from (-)-DMDI to (+)-6a-HMK are unknown.The shifting of the stereochemistry from (-)-DMDI to (+)-6a-HMK has been proposed to involve the achiral isoflavene, 7, 2'-dihydroxy-4', 5'-methylene-dioxyisoflav-3-ene (DMDIF). In this dissertation, I have shown that cis-(-)-DMDI is the enzymatic product of (-)-sophorol, and is the precursor of DMDIF which is produced by the dehydration activity of "isoflavene synthase" (IFVS). IFVS activity was not observed in elicited tissues of alfalfa, chickpea, beans, pepper, and broccoli, plants that do not produce (+) pterocarpans. Partial purification of IFVS demonstrated that it is either large in size or tightly complexed with other proteins. The SDS-PAGE of the 29-fold purified product revealed 12 major bands that aggregated into 3 bands in the non-denaturing PAGE. IFVS activity was in band 3 which co-migrated with marker proteins of>100 kDa in size. Proteins identified from LC-MS/MS peptide sequences of the proteins in band 3, when compared to three protein databases, did not identify any proteins with an enzymatic activity expected for IFVS. A disease resistance-response protein (a dirigent-like protein) and two protein-binding proteins were the most abundantly detected proteins in the pea transcriptome-translated database. Also, four of the known enzymes (isoflavone reductase, HMM1, HMM2, and sophorol reductase) involved in (+)-pisatin biosynthesis were among the proteins identified. It may be that IFVS is associated with these other proteins as a complex in vitro and in vivo. The lack of detection of IFVS in the databases could be because it has not yet been sequenced as it functions in a rare biosynthetic pathway.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectIsoflavonoiden_US
dc.subjectPeaen_US
dc.subjectPhytoalexinen_US
dc.subjectPisatinen_US
dc.subjectPisum sativum Len_US
dc.subjectPlant Pathologyen_US
dc.subjectIsoflaveneen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePlant Pathologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorVanEtten, Hans D.en_US
dc.contributor.committeememberHawes, Martha C.en_US
dc.contributor.committeememberRay, Dennis T.en_US
dc.contributor.committeememberMolnar, Istvanen_US
dc.contributor.committeememberPierson, Leland S.en_US
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