Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo

J Biol Chem. 2004 May 7;279(19):19551-8. doi: 10.1074/jbc.M401261200. Epub 2004 Feb 21.


IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry
  • Amino Acid Sequence
  • Binding Sites
  • Blotting, Western
  • Carbon-Sulfur Lyases / chemistry*
  • Carbon-Sulfur Lyases / genetics
  • Cell Division
  • Chromatography, High Pressure Liquid
  • Cysteine / chemistry
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Hydro-Lyases / chemistry
  • Iron-Sulfur Proteins / chemistry*
  • Models, Chemical
  • Molecular Sequence Data
  • Mutation
  • Phenotype
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA, Transfer / metabolism
  • Subcellular Fractions / metabolism
  • Succinate Dehydrogenase / chemistry
  • Thionucleotides / biosynthesis*


  • Escherichia coli Proteins
  • Iron-Sulfur Proteins
  • IscU protein, E coli
  • Thionucleotides
  • RNA, Transfer
  • Succinate Dehydrogenase
  • Hydro-Lyases
  • phosphogluconate dehydratase
  • Carbon-Sulfur Lyases
  • cysteine desulfurase
  • Cysteine
  • Alanine