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Antioxidant reductive systems in plants : The poplar glutaredoxins C1 and C4. Why genetic engineering and protein structure are a necessary complement.

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Presentation on theme: "Antioxidant reductive systems in plants : The poplar glutaredoxins C1 and C4. Why genetic engineering and protein structure are a necessary complement."— Presentation transcript:

1 Antioxidant reductive systems in plants : The poplar glutaredoxins C1 and C4. Why genetic engineering and protein structure are a necessary complement to Phylogenomics. Jean-Pierre Jacquot UMR 1136 INRA UHP Interaction Arbres Microorganismes IFR 110 Génomique, Ecophysiologie et Ecologie fonctionnelles Université Henri Poincaré, Nancy I, France Décembre 2006 Ecole Phylogénomique Carry Le Rouet Nter Cter GSH C27

2 What is a glutaredoxin? Small oxidoreductases cousin to thioredoxins Generally small molecular weight (ca 12 kDa, 100 to 120 amino acids in the processed form) High degree of secondary structures. Central pleated sheet surrounded by helices. Often thermostable Active site generally of the CXXC form (most well known YCPYC) thioredoxin is generally WCGPC and PDI WCGHC Reduced via glutathione Function : reducers of disulfide bridges, glutathionylation/ de, electron donors to peroxidases, domain of APS reductase Nter Cter GSH C27

3 The cytosolic and mitochondrial pathways leading to reduction of disulfide bonds NADPH SH NTR Trx h,o Thioredoxin pathway HS S S Target protein Target protein Active form Inactive form SH GR Grx SH 2 GSSG GSH GSH/Glutaredoxin pathway SH Nter Cter GSH C27

4 SH GSH GSSG GR Grx GR S S NADP + NADPH S-S- SH S-S- Grx S-SG GSH GSSG SH S-SG Protein ROS Monothiol pathway Dithiol pathway S S Grx Protein SH HS Protein S-S Protein HS Grx S S Catalytic mechanisms of glutaredoxins Nter Cter GSH C27

5 0.1 human Grx1 E. coli Grx 3 human Grx2 E. coli Grx1 At Grx C4 At Grx C3 At Grx C5 At GrxC1 At Grx C2yeast Grx1 yeast Grx2 E. coli Grx4 yeast Grx 5 At Grx S15 human Grx3 At Grx S14 yeast Grx 4 yeast Grx 3 At Grx S1 At Grx S2 At GrxS3 At GrxC12 At Grx C7 At Grx C8 At Grx C13 At Grx C14 At Grx C9 At Grx C10 Cxx[C/S] CGFS CCx[C/S/G] There are active site variants and subgroups of glutaredoxin in all known genomes Rouhier et al. Cell Mol Life Sci Jun;61(11): Nter Cter GSH C27

6 CxxS14 CxxS15 CxxS17 CxxS16 CxxC1 CxxC2 CxxS12 CxxC5 CxxC4 CxxC3 CxxS11 CxxC8 CxxC11 CxxC12 CxxC13 CxxC14 CxxS9 CxxC9 CxxS13 CxxC10 CxxC6 CxxS1 CxxS6 CxxS10 CxxC7 CxxS2 CxxS8 CxxS3 CxxS5 CxxS4 CxxS7 CGFS CCx[C/S/G] Cxx[C/S] There are 31 glutaredoxin genes in A. thaliana, and a similar number in poplar Rouhier et al J Exp Bot. 2006;57(8): Nter Cter GSH C27

7 Characterization of poplar glutaredoxins of the Cxx[C/S] group Glutaredoxins C4 and C1 Nter Cter GSH C27

8 Nter Cter GSH C27 Glutaredoxins C1 and C4 are present in all sequenced plants. They display extensive homologies

9 Glutaredoxin C4 has a CPYC active site What are its targets? What functions can it have? What is its 3D structure? What do we know about the molecular contacts with established targets of known 3D structure? Nter Cter GSH C27

10 Plant glutaredoxin C4 targets Rouhier et al., 2005, Antiox & Red. Signal., 2005 Jul-Aug;7(7-8): Nter Cter GSH C27

11 ROOH ROH Prx SH Prx SOH SH In vitro reduction systems 2 GSH GSSG S Grx S SH Grx SH NADP NADPH,H + GR NADPH,H + NADP NTR S Trx S SH Trx SH ROOH ROH Prx SH Prx SOH SH Rouhier et al. J Biol Chem Apr 19;277(16): Nter Cter GSH C27

12 Nter Cter C51 C76 Nter Cter GSH C27 Poplar type II peroxiredoxin Poplar glutaredoxin C4 Echalier et al. Biochemistry Feb 15;44(6): Corbier et al. unpublished Nter Cter GSH C27 X ray structures

13 3D structure of the Haemophilus influenzae Prx Grx hybrid enzyme Kim et al, J Biol Chem Mar 21;278(12): Nter Cter GSH C27

14 Noguera et al. J Mol Biol Oct 28;353(3): Nter Cter GSH C27 NMR structure of poplar glutaredoxin C4

15 Glutaredoxin C1 has a CGYC active site What is its 3D structure? What are its targets? What functions can it have? What do we know about the molecular contacts with established targets of known 3D structure?

16 When expressed in E.coli cells, poplar glutaredoxin C1 is distributed into two fractions of approximately equal importance One with a monomeric apoprotein (ca 12 kda) One with a dimeric holoprotein (apparent mass 24 kDa) The dimeric holoprotein faction is reddish brown suggesting the presence of an ironsulfur centre Chemical analyses indicate that a dimer contains one 2Fe-2S center

17 Spectral properties of poplar glutaredoxin C1 holo Rouhier et al. unpublished Nter Cter GSH C27

18 X ray 3D structure of poplar Grx C1 Rouhier et al. unpublished Nter Cter GSH C27

19 Feng et al. Biochemistry Jul 4;45(26): Nter Cter GSH C27 NMR structure of poplar Grx C1

20 Both NMR spectroscopy and X ray crystallography indicate that the iron sulfur centre of poplar glutaredoxin C1 bridges two identical subunits The ligands are the active site catalytic cysteine and external molecules of reduced glutathione The backup cysteine or other conserved cysteines in various C1 glutaredoxins are not involved in the binding of the ISC

21 GSH GSSG Mitochondrial CGFS Cytosol C1 CGYC GRX Aconitase SDH ROS sensor? CYTOSOL MITOCHONDRIA Is glutaredoxin C1 involved in the ISC assembly of cytosolic iron sulfur containing enzymes?

22 Conclusion Glutaredoxins are likely to play multiple functions in plant cells Donors for peroxide reduction (interaction with type II peroxiredoxin) Electron donors to APS reductase Possibly implicated in iron sulfur assembly processes (yeast Grx 5 is involved in these pathways) The implication of certain Grx isoforms in the ISC assembly could be related to their own capacity to assemble ISC centers. There are reports that some Grx are also involved/required for heme assembly

23 Collaborations X ray crystallography A Aubry, C Corbier, Nancy, France T Hase, M Kusunoki, Osaka, Japan NMR spectroscopy JM Lancelin, I Krimm, Lyon, France B Xia, Beijing, China RAMAN, EPR spectroscopy M Johnson, Georgia, USA


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