Details
Original language | English |
---|---|
Article number | 668897 |
Journal | Frontiers in Plant Science |
Volume | 12 |
Publication status | Published - 30 Jun 2021 |
Externally published | Yes |
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
- Agricultural and Biological Sciences(all)
- Plant Science
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In: Frontiers in Plant Science, Vol. 12, 668897, 30.06.2021.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
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
Y1 - 2021/6/30
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 -