Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism

Ghulam Hasnain, Jeffrey C. Waller, Sophie Alvarez, Geoffrey E. Ravilious, Joseph M. Jez, Andrew D. Hanson

Research output: Contribution to journalArticle

Abstract

Proteins of the YgfZ family occur in all domains of life and are characterized by the conserved dodecapeptide motif KGC[Y/F]-x-GQE-x 3-[R/K]. YgfZ proteins are known to participate in assembly or repair of iron/sulphur clusters, and to require folate for biological activity, but their mechanism of action is unknown. To assess the importance of individual residues in the conserved motif, Escherichia coli Ygf Z was expressed from a plasmid in a ΔygfZ strain and subjected to alanine-scanning mutagenesis. The impacts on YgfZ functionality were evaluated by assays of growth and of the in vivo activity of the iron/sulphur enzyme MiaB, which modifies tRNA. By these criteria, the motif's tyrosine residue (Y229) had a detectable influence but only the cysteine residue (C228) was critical, for only the C228A mutant failed to complement the growth and MiaB activity phenotypes of the ΔygfZ strain. Immunoblots confirmed that the latter result was not simply because of a low level of the C228A mutant protein. Collectively, these data demonstrate a pivotal role for the Ygf Z motif's cysteine residue and a subsidiary one for the adjacent tyrosine, and help formulate a hypothesis about the folate requirement of Ygf Z proteins.

Original languageEnglish (US)
Pages (from-to)168-172
Number of pages5
JournalFEMS Microbiology Letters
Volume326
Issue number2
DOIs
StatePublished - Jan 1 2012

Fingerprint

Folic Acid
Sulfur
Iron
Cysteine
Tyrosine
Proteins
Mutant Proteins
Growth
Transfer RNA
Mutagenesis
Alanine
Plasmids
Escherichia coli
Phenotype
Enzymes

Keywords

  • Essential cysteine
  • Folate
  • Iron/sulphur proteins
  • MiaB
  • YgfZ

ASJC Scopus subject areas

  • Microbiology
  • Genetics
  • Molecular Biology

Cite this

Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism. / Hasnain, Ghulam; Waller, Jeffrey C.; Alvarez, Sophie; Ravilious, Geoffrey E.; Jez, Joseph M.; Hanson, Andrew D.

In: FEMS Microbiology Letters, Vol. 326, No. 2, 01.01.2012, p. 168-172.

Research output: Contribution to journalArticle

Hasnain, Ghulam ; Waller, Jeffrey C. ; Alvarez, Sophie ; Ravilious, Geoffrey E. ; Jez, Joseph M. ; Hanson, Andrew D. / Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism. In: FEMS Microbiology Letters. 2012 ; Vol. 326, No. 2. pp. 168-172.
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