Download presentation
Presentation is loading. Please wait.
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 Nter Cter GSH C27 Décembre 2006 Ecole Phylogénomique Carry Le Rouet
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 b sheet surrounded by a 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 Target protein Active form Inactive form SH GR Grx 2 GSSG GSH GSH/Glutaredoxin pathway Nter Cter GSH C27
4
Catalytic mechanisms of glutaredoxins
S-SG GSH GSSG SH Protein ROS Monothiol pathway SH GSH GSSG GR Grx S NADP+ NADPH S- Dithiol pathway S Grx Protein SH HS S-S Nter Cter GSH C27
5
There are active site variants and subgroups
yeast Grx 4 CGFS yeast Grx 3 At Grx S14 human Grx3 At Grx S1 CCx[C/S/G] At Grx S2 At GrxS3 At Grx S15 At GrxC12 At Grx C7 At Grx C8 yeast Grx 5 At Grx C13 At Grx C14 E. coli Grx4 At Grx C9 E. coli Grx 3 At Grx C10 human Grx1 human Grx2 yeast Grx2 E. coli Grx1 yeast Grx1 At Grx C2 At Grx C4 At GrxC1 At Grx C3 Cxx[C/S] At Grx C5 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 0.1
6
There are 31 glutaredoxin genes in A. thaliana,
CxxC7 CxxC8 CCx[C/S/G] CxxC11 CxxS4 CxxS10 CxxC12 CxxS6 CxxS8 CxxS7 CxxC13 CxxS5 CxxC14 CxxS3 CxxS2 CxxS9 CxxS1 CxxS11 CxxC6 CxxC1 CxxC10 CxxC2 CxxS12 CxxS13 CxxC5 CxxC9 Cxx[C/S] CxxC4 CxxC3 CxxS17 CxxS15 CxxS16 CxxS14 CGFS 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
Glutaredoxins C1 and C4 are present in all sequenced plants.
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
In vitro reduction systems
Prx SH SOH 2 GSH GSSG S Grx SH NADP NADPH,H+ GR ROOH ROH NADPH,H+ NADP NTR S Trx SH Prx SH SOH ROOH ROH Nter Cter GSH C27 Rouhier et al. J Biol Chem Apr 19;277(16):
12
Poplar type II peroxiredoxin
X ray structures Nter Cter C51 C76 Nter Cter GSH C27 Poplar type II peroxiredoxin Poplar glutaredoxin C4 Nter Cter GSH C27 Echalier et al. Biochemistry Feb 15;44(6): Corbier et al. unpublished
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
NMR structure of poplar glutaredoxin C4
Nter Cter GSH C27 Noguera et al. J Mol Biol Oct 28;353(3):
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
Nter Cter GSH C27 Spectral properties of poplar glutaredoxin C1 holo Rouhier et al. unpublished
18
X ray 3D structure of poplar Grx C1
Nter Cter GSH C27 X ray 3D structure of poplar Grx C1 Rouhier et al. unpublished
19
Feng et al. Biochemistry. 2006 Jul 4;45(26):7998-8008.
NMR structure of poplar Grx C1 Nter Cter GSH C27 Feng et al. Biochemistry Jul 4;45(26):
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
ROS sensor? GRX CYTOSOL GRX ROS sensor? MITOCHONDRIA Aconitase GSH
GSSG Cytosol C1 CGYC CYTOSOL Mitochondrial CGFS GRX SDH ROS sensor? 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
Similar presentations
