The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features

Research output: Contribution to journalArticleResearchpeer review

Authors

  • Adrien Favier
  • Pierre Gans
  • Elisabetta Boeri Erba
  • Luca Signor
  • Soumiya Sankari Muthukumar
  • Thomas Pfannschmidt
  • Robert Blanvillain
  • David Cobessi

External Research Organisations

  • University Grenoble-Alpes (UGA)
  • École supérieure de physique et de chimie industrielles de la ville de Paris (ESPCI)
  • BIOGER INRA Institute
  • Interdisciplinary Research Institute of Grenoble (IRIG)
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Details

Original languageEnglish
Article number668897
JournalFrontiers in Plant Science
Volume12
Publication statusPublished - 30 Jun 2021
Externally publishedYes

Abstract

In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

Keywords

    NMR, X-ray crystallography, chloroplast biogenesis, iron superoxide dismutase, plastid-encoded RNA polymerase

ASJC Scopus subject areas

Cite this

The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features. / Favier, Adrien; Gans, Pierre; Boeri Erba, Elisabetta et al.
In: Frontiers in Plant Science, Vol. 12, 668897, 30.06.2021.

Research output: Contribution to journalArticleResearchpeer review

Favier A, Gans P, Boeri Erba E, Signor L, Muthukumar SS, Pfannschmidt T et al. The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features. Frontiers in Plant Science. 2021 Jun 30;12:668897. doi: 10.3389/fpls.2021.668897
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title = "The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features",
abstract = "In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.",
keywords = "NMR, X-ray crystallography, chloroplast biogenesis, iron superoxide dismutase, plastid-encoded RNA polymerase",
author = "Adrien Favier and Pierre Gans and {Boeri Erba}, Elisabetta and Luca Signor and Muthukumar, {Soumiya Sankari} and Thomas Pfannschmidt and Robert Blanvillain and David Cobessi",
note = "Funding Information: The diffraction experiments were conducted on beamline FIP-BM30A and ID23-1 at the ESRF (Grenoble, France). We thank the beamline staff for technical help, Auriane Bron and Florence Prunier-Bossion for their technical assistance. Funding. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). This work was supported by the Agence National de la Recherche (ANR-17-CE11-0031). ",
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T1 - The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features

AU - Favier, Adrien

AU - Gans, Pierre

AU - Boeri Erba, Elisabetta

AU - Signor, Luca

AU - Muthukumar, Soumiya Sankari

AU - Pfannschmidt, Thomas

AU - Blanvillain, Robert

AU - Cobessi, David

N1 - Funding Information: The diffraction experiments were conducted on beamline FIP-BM30A and ID23-1 at the ESRF (Grenoble, France). We thank the beamline staff for technical help, Auriane Bron and Florence Prunier-Bossion for their technical assistance. Funding. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). This work was supported by the Agence National de la Recherche (ANR-17-CE11-0031).

PY - 2021/6/30

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N2 - In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

AB - In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

KW - NMR

KW - X-ray crystallography

KW - chloroplast biogenesis

KW - iron superoxide dismutase

KW - plastid-encoded RNA polymerase

UR - http://www.scopus.com/inward/record.url?scp=85110172076&partnerID=8YFLogxK

U2 - 10.3389/fpls.2021.668897

DO - 10.3389/fpls.2021.668897

M3 - Article

C2 - 34276730

VL - 12

JO - Frontiers in Plant Science

JF - Frontiers in Plant Science

SN - 1664-462X

M1 - 668897

ER -

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