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Biogenesis of [Fe-S] proteins Frédéric Barras, LCB, CNRS, Marseille Some [Fe-S] clusters Kiley and Beinert, 2003 NIF iscAnif ISC iscR SUF CSD sufA nifU iscS sufB csdA nifS iscU sufC iscA sufD ygdK hscB sufS hscA fdx sufE Three machineries in E. coli ISC SUF CSD iscR sufA iscS sufB csdA iscU sufC iscA sufD ygdK hscB sufS hscA fdx sufE Cysteine S Alanine donor Fe S acceptor S Fe-S Scaffold Fe-S Target SUF machinery Cysteine S donor S Alanine Fe acceptor S Fe-S Scaffold ABC ATPase ? Fe-S Target ISC machinery Cysteine S donor S Alanine Ferredoxin ? acceptor Fe-S Chaperone Fe-S Scaffold Fe-S Target Fe Cysteine S Alanine CSD machinery donor Fe S acceptor S Fe-S Target Substrate specificity Suf Isc Iron assimilation Thionucleosides Fe-S Thiamin Molybdopterin Csd Environmental specificity Suf Isc H2O2 IscR [2Fe-2S] OxyR Fur Csd Nachin et al., 2001,, Schwartz et al., 2001, Zheng et al., 2001, Hantke, 2002 Lee et al., 2004 Biogenesis of [Fe-S] proteins in Cenibacterium Phenotypic complementation E. coli iscS and sufS mutants Library Cenibacterium iscS of Cenibacterium Complementation: thiamine and nicotinic acid requirement Cloned DNA Genome analysis iscR iscS iscU iscA hscB hscA fdx 52 % 69 % 82 % 67 % 39 % 59 % 72 % Genetic organisation identical to E. coli Characterisation of IscSCeni Purification (His)6-IscSCeni IscSCeni Cysteine desulfurase activity PLP/protein µmol alanine/min-1 mig-1 IscScoli IscSCeni CsdAcoli 0.22 0.33 0.5 1 1 1 No SUF machinery in Cenibacterium Complementation : no sufS-containing insert Genome analysis : one cysteine desulfurase = IscS E. coli sufS mutant complementation (Growth in presence of iron chelator ) Cloned DNA Peptidase sodC orf1 cysA cysW 47% id. Cbl 45 % id. CysB Sulfur metabolism regulator cysT Carboxynorspermidine dehydrogenase 10,0 sufS/pcDNA 2.1 0 µM DIP Log (OD 600) sufS/pcDNA 2.1 160 µM DIP sufS/pcDNA 19 160 DIP 1,0 0,1 0 5 10 15 Temps (h) 20 25 30 One machinery in Cenibacterium ISC iscR iscS iscU iscA hscB hscA fdx One machinery in Cenibacterium ISC iscR iscS iscU iscA hscB hscA Implication of the ISC machinery in arsenic resistance Model to study Scaffold proteins Problems to construct mutants……. iscS, iscA, msrA fdx Frédéric Barras Béatrice Py Gabriel Rincón Enríquez Collaborators CEA, Grenoble Sandrine Ollagnier de Choudens Marc Fontecave
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