Antioxidant reductive systems in plants :Antioxidant reductive systems in plants :The poplar glutaredoxins C1 and C4.The poplar glutaredoxins C1 and C4.
Why genetic engineering and protein structure are a Why genetic engineering and protein structure are a necessary complement to Phylogenomics.necessary complement to Phylogenomics.
Jean-Pierre Jacquot
UMR 1136 INRA UHP Interaction Arbres MicroorganismesIFR 110 Génomique, Ecophysiologie et Ecologie fonctionnelles
Université Henri Poincaré, Nancy I, France
Décembre 2006 Ecole Phylogénomique Carry Le Rouet
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What is a glutaredoxin?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
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The cytosolic and mitochondrial pathways The cytosolic and mitochondrial pathways leading to reduction of disulfide bondsleading to reduction of disulfide bonds
NADPH
SHSH
SHSH
NTR
Trx h,o
Thioredoxin pathway
HSHS
SS
Target protein
Target protein Active form
Inactive form
SH SH
GR
Grx
SH
2
GSSG
GSH
GSH/Glutaredoxin pathway
SHSH
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SH
GSH
GSSG
GR Grx
GR
SS
NADP+
NADPH
S-
SH
SH
S-
Grx
S-SG
GSH
GSSGSH
S-SGProtein
Protein
ROS
Monothiol pathway
Dithiol pathway
S
S
Grx
Protein
SH
HSProtein
S-S
ProteinHS
HS
GrxS
S
Catalytic mechanisms of glutaredoxinsCatalytic mechanisms of glutaredoxins
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human Grx1
E. coli Grx 3
human Grx2
E. coli Grx1At Grx C4
At Grx C3
At Grx C5
At GrxC1At 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 S1At Grx S2
At GrxS3At GrxC12
At Grx C7
At Grx C8At Grx C13At Grx C14
At Grx C9
At Grx C10
Cxx[C/S]
CGFS
CCx[C/S/G]
There are active site variants and subgroups There are active site variants and subgroups of glutaredoxin in all known genomesof glutaredoxin in all known genomes
Rouhier et al. Cell Mol Life Sci. 2004 Jun;61(11):1266-77.
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CxxS14
CxxS15
CxxS17
CxxS16
CxxC1
CxxC2
CxxS12
CxxC5
CxxC4
CxxC3
CxxS11
CxxC8
CxxC11
CxxC12
CxxC13CxxC14
CxxS9
CxxC9
CxxS13
CxxC10
CxxC6
CxxS1
CxxS6
CxxS10
CxxC7
CxxS2
CxxS8
CxxS3CxxS5
CxxS4CxxS7
CGFS
CCx[C/S/G]
Cxx[C/S]
There are 31 There are 31 glutaredoxinglutaredoxin genes in A. thaliana, genes in A. thaliana, and a similar number in poplarand a similar number in poplarRouhier et al J Exp Bot. 2006;57(8):1685-96.
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Characterization of poplar glutaredoxins Characterization of poplar glutaredoxins
of the Cxx[C/S] groupof the Cxx[C/S] group
Glutaredoxins C4 and C1Glutaredoxins C4 and C1
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Glutaredoxins C1 and C4 are present in all sequenced plants. Glutaredoxins C1 and C4 are present in all sequenced plants. They display extensive homologiesThey display extensive homologies
Glutaredoxin C4 has a CPYC active siteGlutaredoxin 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?
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Name and functional class Localizationa Peptidesb Homolog Atc Cysteinesd Trx targetse Glutathiolationf Stress related reactions Peroxiredoxin IIF2 M 35 At3g06050 2 Glutathione reductase1 P, M 11 At3g54660 4 Glutaredoxin5 P 15 At2g20270 2 Formate dehydrogenase3 M 13 At5g14780 3 Aldehyde dehydrogenase3 M 13 At3g48000 5 Photorespiration Glycine decarboxylase protein H1 M 11 At1g32470 1 Balmer Glycine decarboxylase protein P1,2 M 11, 22 At4g33010 11 Balmer Translation Glycyl tRNA synthetase1 M 11 At1g29880 10 Heat Shock Protein 70 kDa3 M 13 At5g09590 3 Chaperonin HSP 603 M 23 At2g33210 4 Citric acid cycle and associated reactions
NAD-dependent malate dehydrogenase1 M 21, 25 At1g53240 3 Balmer, maeda yes
Isocitrate dehydrogenase NADP1 M 11 At5g14590 6 Balmer Malic enzyme M 35 At2g13560 2 succinyl CoA ligase beta subunit3 M 13 At2g20420 Respiratory pathway ATP Synthase beta chain3 M 93 At5g08670 2 Balmer ATP Synthase Alpha chain3 M 23 At2g07698 5 Balmer NADH ubiquinone oxidoreductase 22.5 kDa, sub E3 M 13 At5g52840 3
Plant glutaredoxin C4 targetsPlant glutaredoxin C4 targets
Rouhier et al., 2005, Antiox & Red. Signal., 2005 Jul-Aug;7(7-8):919-29. Nter
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ROOH
ROH
PrxSH
SH
PrxSOH
SH
In vitro reduction systemsIn vitro reduction systems
2 GSH
GSSG
S
GrxS
SHGrx
SH
NADP
NADPH,H+
GR
NADPH,H+
NADP
NTR
S
TrxS
SHTrx
SH
ROOH
ROH
PrxSH
SH
PrxSOH
SH
Rouhier et al. J Biol Chem. 2002 Apr 19;277(16):13609-14.
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C76 Nter
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Poplar type II peroxiredoxin Poplar glutaredoxin C4
Echalier et al. Biochemistry. 2005 Feb 15;44(6):1755-67Corbier et al. unpublished
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X ray structuresX ray structures
3D structure of the Haemophilus influenzae 3D structure of the Haemophilus influenzae Prx Grx hybrid enzymePrx Grx hybrid enzymeKim et al, J Biol Chem. 2003 Mar 21;278(12):10790-8.
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Noguera et al. J Mol Biol. 2005 Oct 28;353(3):629-41.
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NMR structure of poplar glutaredoxin C4NMR structure of poplar glutaredoxin C4
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?
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
Spectral properties of poplar glutaredoxin C1 holoSpectral properties of poplar glutaredoxin C1 holoRouhier et al. unpublished
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X ray 3D structure of poplar Grx C1X ray 3D structure of poplar Grx C1Rouhier et al. unpublished
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Feng et al. Biochemistry. 2006 Jul 4;45(26):7998-8008.
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NMR structure of poplar Grx C1NMR structure of poplar Grx C1
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 moleculesof reduced glutathione
The backup cysteine or other conserved cysteines in various C1 glutaredoxins are not involved in the binding of the ISC
GSH
GSSG
Mitochondrial CGFS
Cytosol C1 CGYC
GRX
GRX
Aconitase
SDH
ROS sensor?
ROS sensor?
CYTOSOL
MITOCHONDRIA
Is glutaredoxin C1 involved in the ISC assembly of cytosolic iron sulfur containing enzymes?
Conclusion 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
CollaborationsCollaborations
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