1. Biogenesis of [Fe-S] proteins in Escherichia coli
Frederic Barras, LCB, CNRS, Marseille
Biogenesis of [Fe-S] proteins in Escherichia coli
Marburg, 21 april 2004

2. Biogenesis of [Fe-S] proteins in Escherichia coli
Background
The IscS system: other’s story
The Suf system: our story
The Csd system: the new story

3. Some [Fe-S] clusters Kiley and Beinert, 2003
Trouver differents types de clusters
Kiley and Beinert, 2003

4. Biological functions of [Fe-S] clusters
Electron transfer
Sulfur transfer
Sensing O2 and derivatives

5. [Fe-S] cluster containing proteins
FNR
PJ Kiley

6. [Fe-S] cluster containing proteins Aconitase/IRE-BP
IRE-binding site not accessible
IRE-BP
No [4Fe-4S] cluster
IRE-binding site accessible

7. [Fe-S] cluster containing proteins
SoxR
[2Fe-2S]2+ [2Fe-2S]2+
-35
-10
[2Fe-2S]3+
[2Fe-2S]3+
-35
-10
soxS mRNA
Pomposiello and Demple, 2001

8. How to make [Fe-S] proteins?
1- Chemist’s answer
Apo-protein
[Fe-S] protein
Fe2+
Na2S
DTT

9. How to make [Fe-S] proteins?
2- Biologist’s answer
Source of Fe and S?
(toxicity)
Assembly of [Fe-S] cluster
(insertion and folding)

10. Biogenesis of [Fe-S] proteins in Escherichia coli
Background
The IscS system: other’s story
The Suf system: our story
The Csd system: the new story

11. Study of nitrogen fixation in Azotobacter vinelandii
Dr. Dean’s laboratory (1993)
Apo-nitrogenase
[4Fe-4S] nitrogenase
Fe2+
Cysteine
DTT
NifS

12. Enzymatic activity of Cysteine desulfurases

13. NifS-like gene clusters
A. vinelandii
A. Vinelandii
(isc)
E. coli
(isc)
iscR nifS iscU iscA hscB hscA fdx
R. prowazekii
hscB hscA fdx
iscS iscS iscU iscA iscA2
NFS ISU1, ISA1, JAC SSQ YAH ARH1
S. cerevisiae
Aconitase ou IRE-BP suivant etat redox
Frazzon and Dean, 2003
Muhlenhoff and Lill, 2000

14. [Fe-S] cluster assembly scafold
Functions of the isc gene products
iscR
iscS
iscU
iscA
hscB
hscA
fdx -
Cysteine desulfurase
[2Fe-2S] Ferredoxin
[2Fe-2S]
[Fe-S] cluster assembly scafold
H2O2
Molecular chaperones

15. Model ADP ATP HscA B Apo IscU Cysteine [Fe-S] IscS S Alanine Cysteine
IscA
[Fe-S]
[Fe-S]
Fdx
Alanine

16. 3 NifS-like in E. coli iscS csdA (CSD) sufS (csdB) location 53.7’
37.8’
63.4’
Cysteine Selenocysteine
0.38
3.1
0.9
6.2
0.02
5.5 I II II
AminotransferaseClass V
SSGSACTS
RXGHHCA + +
Structure 3D

17. Loci containing NifS homologues in E. coli
iscR
iscS
iscU
iscA
sufA
sufB
sufC
sufD
sufS
sufE
csdA
ygdK
ygdL

18. Biogenesis of [Fe-S] proteins in Escherichia coli
Background
The IscS system: other’s story
The Suf system: our story
The Csd system: the new story

19. Erwinia chrysanthemi Enterobacteria Plant pathogen
Search for virulence genes by transposon mutagenesis
Beaulieu and van Gijsegem, J Bact, 1990

20. Patzer and Hantke, J Bact 1999, Nachin et al., Mol. Microbiol. 2001
pin10: suf genes
RT-PCR C PCR
sufA
sufB
sufC
sufD
sufS
sufE ‚ ƒ ‚ ƒ
wt - FeSO4
fur - FeSO4
2000
fur + FeSO4
1500
b-glucuronidase actvity
1000
500
wt + FeSO4
200
400
600
800
min
Fur regulated operon
Patzer and Hantke, J Bact 1999, Nachin et al., Mol. Microbiol. 2001

21. Functional Prediction
sufA
sufB
sufC
sufD
sufS
sufE
IscA
Cluster [Fe-S]
formation
SufB
SufD
NifS-like
Cysteine
desulfurase ?
Signatures
ABC ATPase ? ?

22. Takahashi et al., JBC, 2002

23. Archeoglobus fulgidus Bacillus subtilis Methanococcus jannaschii
sufA
sufB
sufC
sufD
sufS
sufE
Erwinia chrysanthemi
Eubacteria
Archaebacteria
Aquifex aeolicus
Archeoglobus fulgidus
Bacillus subtilis
Methanococcus jannaschii
Chlamidiae pneumoniae
M. thermoautotrophicum -
Chlamidiae trachomatis
Pyrococcus abyssi -
Deinococcus radiodurans
Pyrococcus horikoshii
Escherichia coli
Mycobacterium tuberculosis
Synechocystis spp.
Thermotoga maritima
Treponema pallidium
Xylella fastidiosa -

24. ABC transporter ? No TM No TM Walker A and B boxes C region sufB sufC
sufD
No TM
No TM
Walker A and B boxes
C region

25. SufC exhibits ATPase activity
4,5 4
3,5 3
Vm: 4.45 mmole min-1
2,5
M . min-1 2
Km: 0.29 mM m
1,5 1
0,5
0,5 1
1,5 2
[ATP] (mM)

26. Interactions SufB-SufC SufD-SufC
Yeast Two-Hybrid
SufB
SufC
SufD
SufB
SufC
SufD
LexA

27. SufC and SufB are cytoplasmic
Total
Peri
Cyto
Mbrs
Total
Peri
Cyto
Mbrs
SufC
(chromosomal)
Ha-SufB
(plasmidic)
SufC
(plasmidic)
Total
Peri
Cyto
Mbrs
Total
Peri
Cyto
Mbrs
MsrA
MsrA
OutF
OutF
Cel5
Cel5

28. Suf C: an unorthodox cytoplasmic ABC ATPase
SufB
SufC
ATP
SufD
ADP
Nachin et al., EMBO J. 2003

29. Fe-S cluster transfer from HoloSufA/IscA to apoBiotin Synthase.X
holoSufA
holoIscA
(Fe2+ and S2-)
Mature BioB
Ollagnier de Choudens et al., JBC 2003

30. Fe-S transfer « en bloc »
ApoBioB was incubated with 5 mM DTT and a two-fold molar excess of either holoSufA (X) or holoIscA () or a four-fold molar excess of Fe2+ and S2- () and increasing concentrations of bathophenantroline. After 30 minutes incubation at 18°C, biotin synthase activity was measured.

31. SufB
SufC
ATP
SufD
ADP
Fe-S
SufA
Apoprotein
[Fe-S]
Protein [Fe-S]

32. Biochemical analysis Vm (units/mg) SufS 0.02 Csd 1.1 IscS 0.38
Units : µmol Ala / min

33. Structural studies Flexible loop Black: IscS from T. maritima
Cys324
Flexible loop
Black: IscS from T. maritima
White: SufS from E. coli
Mihara, H. et al. (2002) J. Biochem. 131,
IscS from E. coli
Cupp-Vickery, JR et al. (2004) J. Mol. Biol. 330,1049.

34. SufS is activated by SufE
Cysteine
SufS+SufE
SufS+SufE
Selenocysteine
SufS
Loiseau et al., JBC 2003

35. Biochemical analysis Vm (units/mg) SufS 0.021 SufS+SufE 0.750
SufS(C369S)
0.0006
SufS(C369S)+SufE
0.001

36. Sulfur transfer from SufS to SufEB C

37. Alanine
Cysteine
SufS-S364-SH
SufS-S364H
SufE-S51H
SufE-S51-SH
DTT
« S2- »

38. Suf : a [Fe-S] insertion machinery
SufB
SufE
SufS
Cysteine
S2-
SufC
ATP
SufD
ADP
Fe-S
SufA
Apoprotein
[Fe-S]
Protein [Fe-S]
Fe 2+ ???

39. Physiological role of Suf in E. coliwt
+PMS
16+/-2
313+/-1
36+/-9
ratio
13%
244%
97%
sufC
+PMS
9+/-2
283+/-3
39+/-6
ratio 6%
228%
111%
Fumarase
Glut Synthase
PGM
126+/-16
138+/-13
128+/-2
124+/-4
37+/-6
35+/-10

40. MM + glycerol
MM + gluconate
gluconate
6PGDH
(Fe-S)
GND

41. Suf protects oxygen labile
Hypothesis
Suf protects oxygen labile
[Fe-S] clusters

42. Iron acquisition in E. chrysanthemi: an essential virulence factor
Fe3+
Chr=chrysobactin

43. Suf and iron acquisition in E. chrysanthemi
MM+dipyridyl
Strains -
+Fe3+chryso
+FeCl 3
cbs 19 1 20 1
sufB cbs 12 1 20 1
sufC cbs 20 1
sufD cbs 20 1
Hypothesis:
Suf is important for iron acquisition
A basis for importance of Suf in virulence ?

44. Biogenesis of [Fe-S] proteins in Escherichia coli
Background
The IscS system: other’s story
The Suf system: our story
The Csd system: the new story?

45. The new story
Csd
ygdK
ygdL
Cysteine desulfurase
SufE-like
ThiF-like

46. Thiamine biosynthesis early steps
ThiF
ATP O SH
ThiS O OH
ThiS O
OAMP
ThiS
ThiF+ThiI+ThiJ
IscS

47. complex Csd/YgdK/YgdL
Molecular analysis
complex Csd/YgdK/YgdL
Csd
YgdK
YgdL

48. Biochemical analysis Vm (units/mg) Csd 1.1 Csd+YgdK 2.5 Csd(C61)+YgdKND

49. Biochemical analysis Sulfur transfer Targets ? ? S YgdL S YgdK
Cysteine
Csd
YgdL
active S
Alanine

50. Perspectives Iron source ? Mechanistic aspects Role of SufBCD ?
Role of HscAB ?
Role of Fdx ?
Role of IscA vs IscU?
…………
Co-translational insertion vs post-translational repair

51. Perspectives
Substrate and/or Environmental specificity of each system ?
Suf
Isc
Csd
[Fe-S]
Enz-SSH
Thiamin Fe S O Fe S Fe S
IscS-S-S
ThiS Fe S

52. Apoprotein
[Fe-S] Protein
Oxidized [Fe-S]
ROS *
[Fe-S]
Suf
Isc

53. Genetic interaction between Isc and Suf
Synthetic lethality
iscS - DsufABCSDE
Outen et al., Mol Mic, 2004
Suppression
iscS - psufABCSDE
Takahashi et al., JBC, 2002
Redundancy?

54. Apoprotein
[Fe-S] Protein
Oxidized [Fe-S]
ROS *
[Fe-S]
Suf
Isc

55. Imminoaspartate + DHAP
Genetic
Suppression
Imminoaspartate + DHAP
Quinolinic acid
NAD
iscRSUA
NAD-
/pcsdAygdK
NAD+
NadA [4Fe-4S]
Redundancy?

56. Apoprotein
[Fe-S] Protein
Oxidized [Fe-S]
ROS *
[Fe-S]
Csd
Suf
Isc

57. Growth under iron limitation
Strains
Growth under iron limitation
(Dipyridyl 0.32mM)
sufA +
sufB -
sufC
sufC + psufCK40A
sufD
sufS
sufE
sufE + psufEC51S
Suf required under iron limitation

58. Strains
Growth under iron limitation
iscS +
sufS + piscS -
csdA
ygdK
sufS + pcsdA-ygdK
Suf is specifically required under iron limitation

59. Substrate specificity
Suf
Isc
Iron assimilation
Thionucleosides
Fe-S
Thiamin
Molybdopterin
Csd

60. Environmental specificity
Suf
Isc
H2O2
OxyR
IscR
[2Fe-2S]
Fur
Csd
Nachin et al., 2001,, Schwartz et al., 2001, Zheng et al., 2001, Hantke, 2002 Lee et al., 2004

61. Sandrine Ollagnier de Choudens
Actors
LCB, Marseille
Laurent Loiseau
Laurence Nachin
Collaborators
INAPG, Paris
Dominique Expert
CEA, Grenoble
Sandrine Ollagnier de Choudens
Marc Fontecave