Open AccessMutational analysis of YgfZ , a folate‐dependent protein implicated in iron/sulphur cluster metabolism
Author(s) -
Hasnain Ghulam,
Waller Jeffrey C.,
Alvarez Sophie,
Ravilious Geoffrey E.,
Jez Joseph M.,
Hanson Andrew D.
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2011.02448.x
Subject(s) - sulfur metabolism , iron–sulfur cluster , metabolism , biochemistry , sulfur , cluster (spacecraft) , chemistry , genetics , biology , enzyme , organic chemistry , computer science , programming language
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, E scherichia coli Ygf Z was expressed from a plasmid in a Δ ygfZ 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 ( Y 229) had a detectable influence but only the cysteine residue ( C 228) was critical, for only the C 228 A mutant failed to complement the growth and MiaB activity phenotypes of the Δ ygfZ strain. Immunoblots confirmed that the latter result was not simply because of a low level of the C 228 A 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.