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Expression, purification and characterization of an iron-sulfur cluster assembly protein, IscU, from Acidithiobacillus ferrooxidans

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Abstract

An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe2S2] cluster could be assembled in apoIscU with Fe2+ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize l-cysteine and Fe2+ to synthesize [Fe2S2] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe2S2] cluster.

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References

  • Adinolfi S, Rizzo F, Masino L, Nair M, Martin SR, Pastore A, Temussi PA (2004) Bacterial IscU is a well folded and functional single domain protein. Eur J Biochem 271:2093–2100

    Article  PubMed  CAS  Google Scholar 

  • Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, Johnson MK (2000) IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Biochemistry 39:7856–7862

    Article  PubMed  CAS  Google Scholar 

  • Beinert H, Holm RH, Munck E (1997) Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 277:653–659

    Article  PubMed  CAS  Google Scholar 

  • Bertini I, Cowan JA, Del Bianco C, Luchinat C, Mansy SS (2003) Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone. J Mol Biol 331:907–924

    Article  PubMed  CAS  Google Scholar 

  • Ding H, Clark RJ (2004) Characterization of iron binding in IscA, an ancient iron-sulphur cluster assembly protein. Biochem J 379:433–440

    Article  PubMed  CAS  Google Scholar 

  • Fu W, Jack RF, Morgan TV, Dean DR, Johnson MK (1994) nifU gene product from Azotobacter vinelandii is a homodimer that contains two identical [2Fe-2S] clusters. Biochemistry 33:13455–13463

    Article  PubMed  CAS  Google Scholar 

  • Ingledew WJ (1982) Thiobacillus ferrooxidans. The bioenergetics of an acidophilic chemolithotroph. Biochim Biophys Acta 683:89–117

    PubMed  CAS  Google Scholar 

  • Johnson MK (1998) Iron-sulfur proteins: new roles for old clusters. Curr Opin Chem Biol 2:173–181

    Article  PubMed  CAS  Google Scholar 

  • Johnson DC, Unciuleac MC, Dean DR (2006) Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii. J Bacteriol 188:7551–7561

    Article  PubMed  CAS  Google Scholar 

  • Kaut A, Lange H, Diekert K, Kispal G, Lill R (2000) Isa1p is a component of the mitochondrial machinery for maturation of cellular iron-sulfur proteins and requires conserved cysteine residues for function. J Biol Chem 275:15955–15961

    Article  PubMed  CAS  Google Scholar 

  • Kiley PJ, Beinert H (2003) The role of Fe–S proteins in sensing and regulation in bacteria. Curr Opin Microbiol 6:181–185

    Article  PubMed  CAS  Google Scholar 

  • Krebs C, Agar JN, Smith AD, Frazzon J, Dean DR, Huynh BH, Johnson MK (2001) IscA, an alternate scaffold for Fe–S cluster biosynthesis. Biochemistry 40:14069–14080

    Article  PubMed  CAS  Google Scholar 

  • Kurihara T, Mihara H, Kato S, Yoshimura T, Esaki N (2003) Assembly of iron-sulfur clusters mediated by cysteine desulfurases, IscS, CsdB and CSD, from Escherichia coli. Biochim Biophys Acta 1647:303–309

    PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Oganesyan N, Shin DH, Jancarik J, Yokota H, Kim R, Kim SH (2005) Structural characterization of an iron-sulfur cluster assembly protein IscU in a zinc-bound form. Proteins 59:875–881

    Article  PubMed  CAS  Google Scholar 

  • Mansy SS, Wu G, Surerus KK, Cowan JA (2002) Iron-sulfur cluster biosynthesis. Thermatoga maritima IscU is a structured iron-sulfur cluster assembly protein. J Biol Chem 277:21397–21404

    Article  PubMed  CAS  Google Scholar 

  • Nishio K, Nakai M (2000) Transfer of iron-sulfur cluster from NifU to apoferredoxin. J Biol Chem 275:22615–22618

    Article  PubMed  CAS  Google Scholar 

  • Pelzer W, Muhlenhoff U, Diekert K, Siegmund K, Kispal G, Lill R (2000) Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins. FEBS Lett 476:134–139

    Article  PubMed  CAS  Google Scholar 

  • Ramelot TA, Cort JR, Goldsmith-Fischman S, Kornhaber GJ, Xiao R, Shastry R, Acton TB, Honig B, Montelione GT, Kennedy MA (2004) Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site. J Mol Biol 344:567–583

    Article  PubMed  CAS  Google Scholar 

  • Rees DC, Howard JB (2003) The interface between the biological and inorganic worlds: iron-sulfur metalloclusters. Science 300:929–931

    Article  PubMed  CAS  Google Scholar 

  • Schwartz CJ, Djaman O, Imlay JA, Kiley PJ (2000) The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli. Proc Natl Acad Sci USA 97:9009–9014

    Article  PubMed  CAS  Google Scholar 

  • Smid O, Horakova E, Vilimova V, Hrdy I, Cammack R, Horvath A, Lukes J, Tachezy J (2006) Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei. J Biol Chem 281:28679–28686

    Article  PubMed  CAS  Google Scholar 

  • Smith AD, Agar JN, Johnson KA, Frazzon J, Amster IJ, Dean DR, Johnson MK (2001) Sulfur transfer from IscS to IscU: the first step in iron-sulfur cluster biosynthesis. J Am Chem Soc 123:11103–11104

    Article  PubMed  CAS  Google Scholar 

  • Sodeoka M, Larson CJ, Chen L, LeClair KP, Verdine GL (1993) A multifunctional plasmid for protein expression by ECPCR: overproduction of the p50 subunit of NF-KB. Bioorg Med Chem Lett 3:1089–1094

    Article  CAS  Google Scholar 

  • Takahashi Y, Nakamura M (1999) Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli. J Biochem (Tokyo) 126:917–926

    CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  PubMed  CAS  Google Scholar 

  • Tokumoto U, Nomura S, Minami Y, Mihara H, Kato S, Kurihara T, Esaki N, Kanazawa H, Matsubara H, Takahashi Y (2002) Network of protein–protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli. J Biochem (Tokyo) 131:713–719

    CAS  Google Scholar 

  • Tokumoto U, Takahashi Y (2001) Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins. J Biochem (Tokyo) 130:63–71

    CAS  Google Scholar 

  • Tong WH, Rouault TA (2006) Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis. Cell Metab 3:199–210

    Article  PubMed  CAS  Google Scholar 

  • Urbina HD, Silberg JJ, Hoff KG, Vickery LE (2001) Transfer of sulfur from IscS to IscU during Fe/S cluster assembly. J Biol Chem 276:44521–44526

    Article  PubMed  CAS  Google Scholar 

  • Wollenberg M, Berndt C, Bill E, Schwenn JD, Seidler A (2003) A dimer of the FeS cluster biosynthesis protein IscA from cyanobacteria binds a [2Fe2S] cluster between two protomers and transfers it to [2Fe2S] and [4Fe4S] apo proteins. Eur J Biochem 270:1662–1671

    Article  PubMed  CAS  Google Scholar 

  • Yamanaka T, Fukumori Y (1995) Molecular aspects of the electron transfer system which participates in the oxidation of ferrous ion by Thiobacillus ferrooxidans. FEMS Microbiol Rev 17:401–413

    Article  PubMed  CAS  Google Scholar 

  • Yang J, Bitoun JP, Ding H (2006) Interplay of IscA and IscU in biogenesis of iron-sulfur clusters. J Biol Chem 281:27956–27963

    Article  PubMed  CAS  Google Scholar 

  • Yang W, Rogers PA, Ding H (2002) Repair of nitric oxide-modified ferredoxin [2Fe-2S] cluster by cysteine desulfurase (IscS). J Biol Chem 277:12868–12873

    Article  PubMed  CAS  Google Scholar 

  • Zheng L, Cash VL, Flint DH, Dean DR (1998) Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J Biol Chem 273:13264–13272

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Basic Research Program of P.R.China (2004CB619204) and National Natural Science Foundation of P.R.China (50621063).

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Authors and Affiliations

  1. Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha, 410083, P. R. China

    Jia Zeng, Wenjie Zhao, Yuandong Liu, Lexian Xia, Jianshe Liu & Guanzhou Qiu

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  1. Jia Zeng
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  2. Wenjie Zhao
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  3. Yuandong Liu
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  4. Lexian Xia
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  5. Jianshe Liu
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Correspondence to Jianshe Liu.

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Zeng, J., Zhao, W., Liu, Y. et al. Expression, purification and characterization of an iron-sulfur cluster assembly protein, IscU, from Acidithiobacillus ferrooxidans . Biotechnol Lett 29, 1965–1972 (2007). https://doi.org/10.1007/s10529-007-9488-1

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  • DOI: https://doi.org/10.1007/s10529-007-9488-1

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