title: Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism.
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.
authors.affiliation: Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, USA.
authors.forename: Ghulam
authors.initials: G
authors.lastname: Hasnain
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Waller
authors.forename: Sophie
authors.initials: S
authors.lastname: Alvarez
authors.forename: Geoffrey E
authors.initials: GE
authors.lastname: Ravilious
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
mesh_terms: D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism; D015870:Gene Expression; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013466:Sulfurtransferases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; C509203:YgfZ protein, E coli; D005492:Folic Acid; C499584:MiaB protein, E coli; D013466:Sulfurtransferases