YgfZ proteins are known to participate in assembly or repair of i

YgfZ proteins are known to participate in assembly or repair of iron/sulphur clusters, and to require folate for biological activity, but their Selleck MG-132 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 CAL-101 nmr 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. Iron/sulphur (Fe/S) clusters are versatile cofactors with roles that include catalysis, electron transport, regulation, sulphur donation and molybdenum trafficking (Johnson et al., 2005; Dos Santos & Dean, 2008). Although Fe/S clusters

are structurally simple, their assembly depends on complex machinery, the components of which are still not fully known (Johnson et al., 2005; Fontecave & Ollagnier de Choudens, 2008). One such component is the Ygf Z (COG0354) protein family, which is found in all domains of life. Ygf Z proteins have a role in assembly or maintenance of a subset of Fe/S proteins that, in Escherichia coli, includes the tRNA modification enzyme MiaB (Ote et al., 2006; Gelling et al., 2008; Waller et al., 2010). Besides reduced

activity of MiaB and other Fe/S enzymes, E. coli ΔygfZ Rolziracetam strains show various phenotypic defects, including slowed growth and sensitivity to the oxidative stress agent plumbagin (Ote et al., 2006; Lin et al., 2010; Waller et al., 2010). Bacterial, animal, protistan and plant Ygf Z proteins have all been shown to require folate for action in vivo (Waller et al., 2010, 2011), but the biochemical basis of this requirement is not understood. It has, however, been shown that the requirement is most probably for tetrahydrofolate itself, rather than for a one-carbon substituted form (Waller et al., 2010). Ygf Z proteins are characterized by the motif KGC[Y/F]-x-GQE-x3-[R/K], of which the arginine/lysine residue initially escaped notice (Teplyakov et al., 2004). The published three-dimensional structure of E. coli Ygf Z places this motif in a surface loop of the monomer, with the cysteine residues (C228) in two molecules linked via a disulphide bridge, forming a YgfZ dimer (Teplyakov et al., 2004).

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