Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

Handle URI:
http://hdl.handle.net/10754/325337
Title:
Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis
Authors:
Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten
Abstract:
Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.
KAUST Department:
KAUST Catalysis Center (KCC)
Citation:
Witz S, Panwar P, Schober M, Deppe J, Pasha FA, et al. (2014) Structure-Function Relationship of a Plant NCS1 Member - Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis. PLoS ONE 9: e91343. doi:10.1371/journal.pone.0091343.
Publisher:
Public Library of Science
Journal:
PLoS ONE
Issue Date:
12-Mar-2014
DOI:
10.1371/journal.pone.0091343
PubMed ID:
24621654
PubMed Central ID:
PMC3951388
Type:
Article
ISSN:
19326203
Appears in Collections:
Articles; KAUST Catalysis Center (KCC)

Full metadata record

DC FieldValue Language
dc.contributor.authorWitz, Sandraen
dc.contributor.authorPanwar, Pankajen
dc.contributor.authorSchober, Markusen
dc.contributor.authorDeppe, Johannesen
dc.contributor.authorPasha, Farhan Ahmaden
dc.contributor.authorLemieux, M. Joanneen
dc.contributor.authorMöhlmann, Torstenen
dc.date.accessioned2014-08-27T09:47:43Zen
dc.date.available2014-08-27T09:47:43Zen
dc.date.issued2014-03-12en
dc.identifier.citationWitz S, Panwar P, Schober M, Deppe J, Pasha FA, et al. (2014) Structure-Function Relationship of a Plant NCS1 Member - Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis. PLoS ONE 9: e91343. doi:10.1371/journal.pone.0091343.en
dc.identifier.issn19326203en
dc.identifier.pmid24621654en
dc.identifier.doi10.1371/journal.pone.0091343en
dc.identifier.urihttp://hdl.handle.net/10754/325337en
dc.description.abstractPlastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.en
dc.language.isoenen
dc.publisherPublic Library of Scienceen
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en
dc.subjectadenineen
dc.subjectcarrier proteinen
dc.subjectguanineen
dc.subjectnucleobase transporteren
dc.subjectnucleobase transporter PLUTOen
dc.subjectprotein MHP 1en
dc.subjectunclassified drugen
dc.subjecturacilen
dc.subjectamino acid transporten
dc.subjectArabidopsisen
dc.subjectbinding siteen
dc.subjectcontrolled studyen
dc.subjectcrystal structureen
dc.subjectenzyme specificityen
dc.subjectMicrobacterium liquefacienen
dc.subjectmolecular dynamicsen
dc.subjectmutagenesisen
dc.subjectmutational analysisen
dc.subjectsequence homologyen
dc.subjectstructure activity relationen
dc.titleStructure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsisen
dc.typeArticleen
dc.contributor.departmentKAUST Catalysis Center (KCC)en
dc.identifier.journalPLoS ONEen
dc.identifier.pmcidPMC3951388en
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDepartment of Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germanyen
dc.contributor.institutionMembrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, AB, Canadaen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.