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• W2149776132 abstract "SufA is a component of the recently discoveredsuf operon, which has been shown to play an important function in bacteria during iron-sulfur cluster biosynthesis and resistance to oxidative stress. The SufA protein from Erwinia chrysanthemi, a Gram-negative plant pathogen, has been purified to homogeneity and characterized. It is a homodimer with the ability to assemble rather labile [2Fe-2S] and [4Fe-4S] clusters as shown by Mössbauer spectroscopy. These clusters can be transferred to apoproteins such as ferredoxin or biotin synthase during a reaction that is not inhibited by bathophenanthroline, an iron chelator. Cluster assembly in these proteins is much more efficient when iron and sulfur are provided by holoSufA than by free iron sulfate and sodium sulfide. We propose the function of SufA is that of a scaffold protein for [Fe-S] cluster assembly and compare it to IscA, a member of theisc operon also involved in cluster biosynthesis in both prokaryotes and eukaryotes. Mechanistic and physiological implications of these results are also discussed. SufA is a component of the recently discoveredsuf operon, which has been shown to play an important function in bacteria during iron-sulfur cluster biosynthesis and resistance to oxidative stress. The SufA protein from Erwinia chrysanthemi, a Gram-negative plant pathogen, has been purified to homogeneity and characterized. It is a homodimer with the ability to assemble rather labile [2Fe-2S] and [4Fe-4S] clusters as shown by Mössbauer spectroscopy. These clusters can be transferred to apoproteins such as ferredoxin or biotin synthase during a reaction that is not inhibited by bathophenanthroline, an iron chelator. Cluster assembly in these proteins is much more efficient when iron and sulfur are provided by holoSufA than by free iron sulfate and sodium sulfide. We propose the function of SufA is that of a scaffold protein for [Fe-S] cluster assembly and compare it to IscA, a member of theisc operon also involved in cluster biosynthesis in both prokaryotes and eukaryotes. Mechanistic and physiological implications of these results are also discussed. iron-sulfur cluster dithiothreitol biotin synthase ferredoxin iron-sulfur cluster pyridoxal 5-phosphate bathophenanthroline disulfonate deazaflavin vesicular somatidis virus-glycoprotein nickel-nitrilotriacetic acid Iron-sulfur [Fe-S] proteins play important roles in electron transfer, in redox and non-redox catalysis, in regulation, and as sensors within all living organisms, prokaryotes and eukaryotes (1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1530) Google Scholar, 2Beinert H. J. Biol. Inorg. Chem. 2000; 5: 2-15Crossref PubMed Scopus (538) Google Scholar). The biosynthetic process by which defined proportions of iron and sulfur atoms are mobilized from their storage sources and combined in a controlled way to generate the various iron-sulfur cluster prosthetic groups is still far from understood. It requires a complex protein machinery that is only now becoming identified and characterized. In the bacteria Escherichia coli and Azotobacter vinelandii, from which most of the available information is derived, this machinery has been found to be encoded by a highly conserved cluster of at least seven genes,iscRSUA-hscBA-fdx, also named the ISC1 (foriron-sulfur cluster) machinery (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar,4Frazzon J. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14751-14753Crossref PubMed Scopus (29) Google Scholar). Disruption of these genes generally results in both decreased cluster content and activity of many important [Fe-S] enzymes such as aconitase or succinate dehydrogenase, whereas overexpression of the operon yields increased production of recombinant [Fe-S] proteins (5Takahashi Y. Nakamura M. J. Biochem. 1999; 126: 917-926Crossref PubMed Scopus (228) Google Scholar, 6Nakamura M. Saeki K. Takahashi Y. J. Biochem. 1999; 126: 10-18Crossref PubMed Scopus (168) Google Scholar, 7Schwartz C.J. Djaman O. Imlay J.A. Kiley P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9009-9014Crossref PubMed Scopus (258) Google Scholar, 8Tokumoto U. Takahashi Y. J. Biochem. 2001; 130: 63-71Crossref PubMed Scopus (215) Google Scholar, 9Skovran E. Down D.M. J. Bacteriol. 2000; 182: 3896-3903Crossref PubMed Scopus (68) Google Scholar). Homologs to the proteins of the isc operon fromE. coli have been found in the mitochondria of eukaryotes and, in yeast, shown by genetic experiments to play crucial roles in [Fe-S] cluster assembly (10Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, 11Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 12Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar, 13Li J. Kogan M. Knight S.A.B. Pain D. Dancis A. J. Mol. Chem. 1999; 274: 33025-33034Scopus (174) Google Scholar, 14Lange H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (247) Google Scholar, 15Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 16Garland S.A. Hoff K.G. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (167) Google Scholar, 17Schilke B. Voisine C. Beinert H. Craig E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10206-10211Crossref PubMed Scopus (268) Google Scholar, 18Voisine C. Schilke B. Ohlson M. Beinert H. Marszalek K. Craig E.A. Mol. Cell. Biol. 2000; 20: 3677-3684Crossref PubMed Scopus (73) Google Scholar, 19Voisine C. Cheng Y.C. Ohlson M. Schilke B. Hoff K. Beinert H. Marszalek J. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1483-1488Crossref PubMed Scopus (112) Google Scholar, 20Lutz T. Westermann B. Neupert W. Herrmann J.M. J. Mol. Biol. 2001; 307: 815-825Crossref PubMed Scopus (117) Google Scholar). IscS proteins are pyridoxal-phosphate (PLP)-dependent cysteine desulfurases that catalyze the mobilization of sulfur atom from cysteine for incorporation into clusters (21Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22Zheng L. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (358) Google Scholar). IscU and IscA proteins are able to assemble transient and labile [2Fe-2S] and [4Fe-4S] clusters, as shown by Mössbauer and Raman resonance spectroscopy (23Ollagnier-de Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 24Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar, 25Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar, 26Krebs C. Agar J.N. Smith A.D. Frazzon J. Dean D.R. Huynh B.H. Johnson M.K. Biochemistry. 2001; 40: 14069-14080Crossref PubMed Scopus (210) Google Scholar, 27Mansy S.S. Wu G. Surerus K.K. Cowan J.A. J. Biol. Chem. 2002; 277: 21397-21404Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Morimoto K. Nishio K. Nakai M. FEBS Lett. 2002; 519: 123-127Crossref PubMed Scopus (36) Google Scholar, 29Wu G. Mansy S.S. Hemann C. Hille R. Surerus K.K. Cowan J.A. J. Biol. Inorg. Chem. 2002; 7: 526-532Crossref PubMed Scopus (70) Google Scholar, 30Wu G. Mansy S.S. Wu S. Surerus K.K. Foster M.W. Cowan J.A. Biochemistry. 2002; 41: 5024-5032Crossref PubMed Scopus (73) Google Scholar, 31Wu S. Wu G. Surerus K.K. Cowan J.A. Biochemistry. 2002; 41: 8876-8885Crossref PubMed Scopus (93) Google Scholar). These clusters can be rather efficiently transferred to apoferredoxins in vitro, and both IscU and IscA were proposed to function as scaffold proteins for mediating general [Fe-S] cluster assembly (23Ollagnier-de Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 27Mansy S.S. Wu G. Surerus K.K. Cowan J.A. J. Biol. Chem. 2002; 277: 21397-21404Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 30Wu G. Mansy S.S. Wu S. Surerus K.K. Foster M.W. Cowan J.A. Biochemistry. 2002; 41: 5024-5032Crossref PubMed Scopus (73) Google Scholar, 31Wu S. Wu G. Surerus K.K. Cowan J.A. Biochemistry. 2002; 41: 8876-8885Crossref PubMed Scopus (93) Google Scholar, 32Nishio K. Nakai M. J. Biol. Chem. 2000; 275: 22615-22618Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). HscA and HscB proteins are molecular chaperones (33Seaton B.L. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2066-2070Crossref PubMed Scopus (83) Google Scholar). They both interact with IscU, and a complex has been detected in two-hybrid experiments between HscA and IscA (34Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 35Hoff K.G. Ta D.T. Tapley T.L. Silberg J.J. Vickery L.E. J. Biol. Chem. 2002; 277: 27353-27359Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 36Silberg J.J. Hoff K.G. Tapley T.L. Vickery L.E. J. Biol. Chem. 2001; 276: 1696-1700Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 37Tokumoto U. Nomura S. Minami Y. Mihara H. Kato S. Kurihara T. Esaki N. Kanazawa H. Matsubara H. Takahashi Y. J. Biochem. 2002; 131: 713-719Crossref PubMed Scopus (97) Google Scholar). As a consequence they are supposed to be required for optimizing conformations that facilitate [Fe-S] cluster assembly and transfer from IscU/IscA to the target apoproteins. IscU but not IscA also makes a complex with IscS from which it directly gets the sulfur atoms required for [Fe-S] cluster synthesis (25Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar, 38Kato S. Mihara H. Kurihara T. Takahashi Y. Tokumoto U. Yoshimura T. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5948-5952Crossref PubMed Scopus (115) Google Scholar, 39Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 40Smith A.D. Agar J.N. Johnson K.A. Frazzon J. Amster I.J. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2001; 123: 11103-11104Crossref PubMed Scopus (173) Google Scholar). Finally, both IscU and IscA can form complexes with Fdx, the product of another gene of the ISC machinery (23Ollagnier-de Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 29Wu G. Mansy S.S. Hemann C. Hille R. Surerus K.K. Cowan J.A. J. Biol. Inorg. Chem. 2002; 7: 526-532Crossref PubMed Scopus (70) Google Scholar, 30Wu G. Mansy S.S. Wu S. Surerus K.K. Foster M.W. Cowan J.A. Biochemistry. 2002; 41: 5024-5032Crossref PubMed Scopus (73) Google Scholar). Fdx is a [2Fe-2S] ferredoxin, thus suggesting that electron transfer steps are part of the cluster assembly process (41Ta D.T. Vickery L.E. J. Biol. Chem. 1992; 267: 11120-11125Abstract Full Text PDF PubMed Google Scholar). Even though all of these proteins have been isolated in pure form and extensively characterized, the detailed mechanism by which they work together to incorporate an [Fe-S] cluster into an apoprotein is not known and is the subject of intense studies in several laboratories. As part of our efforts to understand this important biological reaction, we studied the properties of the IscA protein from E. coli (23Ollagnier-de Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). There are many reasons for this. First, it is important to understand why most organisms contain both IscA and IscU proteins, with apparently similar functions. It is generally assumed that IscU proteins are the key players in the cluster assembly process, even though there is no clear evidence for it. In support to this notion is the observation that, in Saccharomyces cerevisiae, a knockout of both iscU homologs, ISU1 andISU2, is lethal, whereas a knockout of both iscAhomologs, ISA1 and ISA2, only results in retarded growth on non-fermentable carbon sources, accumulation of iron in mitochondria, and marked decrease in the activities of mitochondrial and cytosolic [Fe-S] enzymes (12Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar, 15Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 16Garland S.A. Hoff K.G. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (167) Google Scholar, 42Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). On the other hand, it was recently shown, using an in vitro [Fe-S] cluster assembly assay, that extracts from S. cerevisiaemitochondria depleted in ISA1 displayed severely decreased activities during cluster assembly in biotin synthase, a [4Fe-4S] enzyme, when compared with wild-type extracts (43Mühlenhoff U. Richhardt N. Gerber J. Lill R. J. Biol. Chem. 2002; 277: 29810-29816Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). A second indication for the importance of IscA-type proteins is the recent discovery of an additional operon in bacteria and intracellular parasites, namedsuf, also involved in iron and sulfur metabolism. Thesuf operon contains six genes, sufABCDSE, including an iscA homolog, sufA, and aniscS homolog, sufS (44Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar, 45Ellis K.E.S. Clough B. Saldanha J.W. Wilson R.J.M. Mol. Microbiol. 2001; 41: 973-981Crossref PubMed Scopus (87) Google Scholar, 46Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). In contrast to S. cerevisiae, the genes of theisc operon are necessary for optimal growth but are not essential for the viability of E. coli cells. In an E. coli mutant, in which the entire isc operon has been deleted, the activity of [Fe-S] proteins is only 2–10% of their activity in wild-type cells (46Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). This is presumably due to the presence of the suf genes as supported by the fact that overexpression of suf operon restores the growth phenotype and activity of [Fe-S] proteins in the mutant cells lacking all components of the ISC machinery. Furthermore, lethality was observed when both the isc and suf operons were inactivated (46Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Genetic experiments using Erwinia chrysanthemi, a Gram-negative plant pathogen, and E. coli, have recently provided more detailed information on the suf genes. In both species the suf operon was found to be under the Fe-dependent repressor Fur, and, in E. coli at least, it was also found to belong to the oxidative stress OxyR-dependent regulon (47Zheng M. Wang X. Templeton L.J. Smulski D.R. laRossa R.A. Storz G. J. Bacteriol. 2001; 183: 4562-4570Crossref PubMed Scopus (660) Google Scholar, 48Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (156) Google Scholar, 49Hantke K. J. Mol. Microbiol. Biotechnol. 2002; 4: 217-222PubMed Google Scholar). This suggests a specific function of the SUF machinery during repair of [Fe-S] clusters as a consequence of oxidative stress and a strong iron limitation. SufC appears to be the most critical protein in this system, because inactivation of sufC in E. chrysanthemi resulted in (i) decreased Fe uptake, (ii) increased accumulation of free intracellular iron levels, (iii) increased sensitivity to oxidative stress, (iv) delay of the induction of the transcriptional activator SoxS by SoxR, a homodimeric [2Fe-2S] redox-regulated transcription factor, (v) decrease activities of enzymes containing oxygen labile [Fe-S] clusters, under oxidative stress, and (vi) decreased virulence of E. chrysanthemi (44Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar, 48Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (156) Google Scholar, 50Nachin L. Loiseau L. Expert D. Barras D. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (221) Google Scholar). Decreased iron uptake was shown to be a consequence of a decreased ability to obtain iron from ferrisiderophores, such as chrysobactin or ferrioxamin, possibly because [Fe-S] clusters in ferrisiderophore reductases are not correctly assembled in this mutant (44Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar, 48Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (156) Google Scholar, 50Nachin L. Loiseau L. Expert D. Barras D. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (221) Google Scholar). With the exception of SufS, the Suf proteins have been very little studied at the biochemical level so far (51Mihara H. Maeda M. Fujii T. Kurihara T. Hata Y. Esaki N. J. Biol. Chem. 1999; 274: 14768-14772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 52Mihara H. Fujii T. Kato K. Hata Y. Esaki N. J. Biochem. 2002; 131: 679-685Crossref PubMed Scopus (61) Google Scholar, 53Lima C.D. J. Mol. Biol. 2002; 315: 1199-1208Crossref PubMed Scopus (83) Google Scholar). However, this system provides a unique tool to understand mechanisms of [Fe-S] cluster assembly, because it is simpler than the ISC system. Indeed, it does not contain equivalents of the ferredoxin protein (Fdx) and of the molecular chaperones (HscA and HscB). As discussed above we were intrigued by the absence of an IscU-type protein, whereas a protein (SufA) displaying significant sequence homology to IscA is part of the machinery. This gives an opportunity to investigate the specific function of an IscA-type protein. Fig. 1 shows the amino acid sequences of SufA proteins from a variety of bacterial and archaeal sources. Alignment with IscA proteins is possible demonstrating 52% identity between SufA and IscA from the same organism, E. coli. In particular, the three invariant cysteines of IscA, which have been shown by site-directed mutagenesis in the case of the yeast protein to be essential for function and were proposed to be involved in iron binding, are present in SufA proteins (15Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 42Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). It is thus very tempting to suppose that SufA has the potential to assemble an [Fe-S] cluster and to serve in a way similar to IscA, as a scaffold protein, for mediating [Fe-S] cluster assembly in target proteins. With this protein the only other protein that SUF shares with ISC is a cysteine desulfurase (SufS), which displays 23% identity to IscS. On the other hand, the suf operon contains proteins that are not present in cluster of ISC genes. The available data suggest that SufB, SufC, and SufD, which are encoded by highly conserved genes occurring in bacteria, archaea, plants, and parasites, work together to form a multimeric ABC transporter complex with ATPase activity (50Nachin L. Loiseau L. Expert D. Barras D. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (221) Google Scholar). The role of the SufBCD complex is still unclear, but a possible function would be to provide energy to the SUF machinery for [Fe-S] cluster assembly. Additional characterization is needed to confirm the presence and the role of the complex. Finally, nothing is known about the function of SufE. In E. coli, a homolog ofsufE, referred as ygdK, lies just after the third cysteine desulfurase of that microorganism, named csd, indicating a conservation of genetic organization betweensufS-sufE and csd-ygdK (48Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (156) Google Scholar). Thus it is tempting to suggest that SufE is also involved in [Fe-S] assembly. Similar examples of this association are found in Pseudomonas aeruginosa, Vibrio cholerae, and Haemophilus influenzae (45Ellis K.E.S. Clough B. Saldanha J.W. Wilson R.J.M. Mol. Microbiol. 2001; 41: 973-981Crossref PubMed Scopus (87) Google Scholar). Here we report, for the first time, the isolation and characterization of SufA protein. We show that SufA from E. chrysanthemi can assemble iron-sulfur clusters, which can be efficiently transferred to target apoproteins. All chemicals were of reagent grade and obtained from Sigma-Aldrich Chemical Co. or Fluka, unless otherwise stated.57Fe2O3 was converted into ferric chloride by dissolving it in hot concentrated (35%) hydrochloric acid of analytical grade (Carlo Erba) and repeatedly concentrated in water. 5-Deaza-7,8-demethyl-10-methylisoalloxazine (DAF) was prepared according to Ashton et al. (54Ashton W.T. Brown R. Tolman R.L. J. Heterocycl. Chem. 1978; 15: 489-491Crossref Scopus (24) Google Scholar).S-Adenosylmethionine was from Roche Applied Science. Pyridoxal 5′-phosphate was from Interchim. IscA, flavodoxin, and flavodoxin reductase were available in our laboratory. Enzymes, oligonucleotides, and culture media were purchased from New England BioLabs, Oligo Express, and Difco, respectively. Pwo DNA polymerase and the High Pure Plasmid Isolation kit, for plasmid DNA purification, were from Roche Diagnostics (Mannheim, Germany). Isolation of DNA fragments from agarose gels was performed using a QIAquick gel extraction kit (Qiagen). DNA sequencing was performed by Genome Express (France). Antibodies against VSV-G were obtained from Roche Applied Science. E. coli MG1655Δsuf was described previously (50Nachin L. Loiseau L. Expert D. Barras D. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (221) Google Scholar). The sufA gene, encoding the SufA protein, was amplified by PCR using genomic E. chrysanthemi 3937 DNA as a template. Primers used for sufA amplification were 5′-CCGCATATGCAAACGCACGATGTAG-3′ (forward primer, underlined bases indicate a NdeI site) and 5′-GTTCTCGAGAAGCCCAAAACTTTCGCC-3′ (reverse primer, underlined bases indicate a XhoI site). PCR was run as follows: genomic DNA was denatured for 10 min at 94 °C. ThePwo DNA polymerase (0.5 unit), deoxynucleotide mix (0.2 mm each), and the primers (0.5 mm final concentration) were added, and 30 cycles (30 s at 94 °C, 30 s at 50 °C, then 30 s at 72 °C) were then performed, followed by a final 10-min elongation step at 72 °C. The PCR product was digested with NdeI and XhoI and then ligated into a pET22b(+) vector (Novagen), digested with the same restriction enzymes, for production of SufA with a histidine tag. The cloned gene was then sequenced to ensure that no error was introduced during PCR reaction. The plasmid was then named pET/SufA. For the cross-linking experiment, a VSV-SufA encoding protein was constructed as follow. Primers used for sufA amplification were 5′-CCCTACCATGGCTTACACTGATATCGAAATGAACCGCCTGGGTAAGATGCAAACGCACGATGTAGA-3′ (forward primer, underlined bases indicate a NcoI site) and 5′-GCCTTCTCGAGTTAAAGCCCAAAACTTTCGC-3′ (reverse primer, underlined bases indicate a XhoI site). PCR was run as follows: genomic DNA was denatured for 2 min at 94 °C. ThePwo DNA polymerase (0.5 unit), deoxynucleotide mix (0.2 mm each), and the primers (0.5 mm final concentration) were added and 30 cycles (30 s at 94 °C, 30 s at 60 °C, then 30 s at 72 °C) were then performed followed by a final 10-min elongation step at 72 °C. The PCR product was digested with NcoI and XhoI and then ligated into pBAD24 vector, digested with NcoI/SalI enzymes. The cloned gene was then sequenced to ensure that no error was introduced during PCR reaction. The plasmid was then named pA-VSV-SufA. For overproduction of SufA, E. coli-competent BL21(DE3) strains were transformed with pET/SufA vector, and 2 × 100 ml overnight cultures were used as an inoculum for 2 × 400 ml of LB medium (Difco) containing 100 μg/ml ampicillin. Cells were grown at 37 °C to an A600 of 0.5, and expression was induced with 0.5 mm of isopropyl-β-d-thiogalactopyranoside (Eurogentec) for 4 h at 37 °C. The bacterial pellet (7 g/800-ml culture) was resuspended in 50 ml of buffer A (100 mm Tris-HCl, pH 7.5, 50 mm NaCl) and treated twice with a French press for disruption. The cell lysate was centrifuged at high speed for 30 min, at 4 °C. The supernatant (180 mg of soluble proteins) was loaded onto a 5-ml Hi-trap column (Amersham Biosciences), charged with nickel, and equilibrated with buffer A. Pure protein (47 mg) was eluted by a 25-ml gradient from 0.04 to 0.5 m imidazole with buffer B (buffer A plus 1 m imidazole) and was washed twice with 10 ml of buffer A onto a BIOMAX-5K device (Millipore) to remove imidazole. SufA protein was then aliquoted and stored at −80 °C. Fast protein liquid chromatography gel filtration with an analytical Superdex-75 (Amersham Biosciences) at a flow rate of 0.5 ml/min equilibrated with buffer A was used for size determination. A gel filtration calibration kit (calibration protein II, Roche Applied Science) was used as the molecular weight standard. E. chrysanthemiA3559 strain was grown overnight in LB medium at 30 °C. When the strain was transformed with plasmid pA-VSV-SufA, ampicillin was added to the medium. A culture was used to inoculate fresh LB medium atA600 0.35. After growth for 1 h,l-arabinose 0.02% (w/v) was added and cultures were incubated for 4 h at 30 °C. Culture was then divided in two equal samples. On one hand, cells were pelleted and resuspended in 1.5 ml of Tris buffer (40 mm, pH 7.5) allowing for the estimation of the amount of Suf proteins present in the cells. On the other hand, spheroplasts were prepared (in 1.5 ml of Tris buffer) as described by Bortoli-German et al. (55Bortoli-German I. Brun E. Py B. Chippaux M. Barras F. Mol. Microbiol. 1994; 11: 545-553Crossref PubMed Scopus (77) Google Scholar). After centrifugation (10,000 rpm, 10 min, 4 °C) periplasmic fractions containing supernatants were stored at 4 °C. Spheroplasts were washed, resuspended in 1.5 ml of Tris buffer (40 mm, pH 7.5), and disrupted with pressure by French press treatment. After centrifugation (15,000 rpm, 15 min, 4 °C), supernatants were submitted to ultracentrifugation at 45,000 rpm during 1.5 h at 4 °C. The resulting supernatants corresponded to cytosol. Membranes were resuspended in 1.5 ml of Tris buffer (40 mm, pH 7.5). VSV-tagged SufA protein was detected using antibody raised against the VSV-G epitope. For each location experiment, efficiency and reliability of the cell fractionation procedure were checked using antibody raised against Cel5, OutF, and MsrA. The following procedure was made anaerobically inside a glove box (Jacomex B553 (NMT)). ApoSufA is obtained by irradiation with DAF in the presence of 10 mm EDTA. After 1 h of incubation, the colorless protein was purified onto a Sephadex G-25 column equilibrated with buffer C (0.1 m Tris-HCl, pH 8.0). ApoSufA (250–500 μm monomer) was then incubated in buffer C for 3–4 h with a 3- to 4-fold molar excess of both Na2S (Fluka) and either Fe(NH4)2(SO4)2(Aldrich) or 57FeCl3 in the presence of 5 mm dithiothreitol (DTT). Then 2 mm EDTA was added, and the solution was further incubated for 30 min. The protein was desalted on Sephadex G-25 (80 ml, same buffer), and the colored fractions were concentrated on Nanosep 10 (Amicon). Reconstitution of apoprotein was achieved with56Fe as described previously (56Ollagnier-de Choudens S. Sanakis Y. Hewitson K. Roach P. Münck E. Fontecave M. J. Biol. Chem. 2002; 277: 13449-13454Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and desalted over Sephadex G-25 (equilibrated with buffer C) to remove adventitiously bound iron after reconstitution. Apoferredoxin was obtained from holoferredoxin as already described (32Nishio K. Nakai M. J. Biol. Chem. 2000; 275: 22615-22618Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Typically, apoferredoxin (76 nmol) was incubated with holoSufA (76 nmol) for 2 h at 25 °C in buffer D (0.1m Tris-HCl, pH 8.0, 30 mm KCl). Transfer of the [Fe-S] cluster from holoSufA to ferredoxin was monitored at time intervals by EPR spectroscopy at liquid helium temperature, from the ferredoxin-characteristic EPR signal obtained after reduction of the cluster with 2 mm dithionite. ApoBioB was obtained by irradiation of the as-isolated enzyme with DAF in the presence of 10 mm EDTA and purified onto a Sephadex G-25 column equilibrated with buffer C. ApoBioB was then incubated in buffer C with either a 2-fold molar excess of holoSufA or a 4-fold molar excess of Fe2+ and S2−. At time intervals (5, 10, 20, 30, and 60 min) biotin synthase activity was measured by addition of all components required for the activity as described below. HoloIscA from E. coli, prepared as previously discussed (23Ollagnier-de Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scho" @default.
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• W2149776132 date "2003-05-01" @default.
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• W2149776132 title "SufA from Erwinia chrysanthemi" @default.
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• W2149776132 doi "https://doi.org/10.1074/jbc.m300285200" @default.
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