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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GSHC27

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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GSHC27

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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Cter

GSHC27

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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GSHC27

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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GSHC27

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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GSHC27

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C51

C76 Nter

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GSHC27

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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GSHC27

Noguera et al. J Mol Biol. 2005 Oct 28;353(3):629-41.

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GSHC27

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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GSHC27

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