title: Identification of altered function alleles that affect Bacillus subtilis PerR metal ion selectivity.
abstract: Bacillus subtilis PerR is a Fur family repressor that senses hydrogen peroxide by metal-catalyzed oxidation. PerR contains a structural Zn(II) ion (Site 1) and a regulatory metal binding site (Site 2) that, upon association with either Mn(II) or Fe(II), allosterically activates DNA binding. In addition, a third less conserved metal binding site (Site 3) is present near the dimer interface in several crystal structures of homologous Fur family proteins. Here, we show that PerR proteins with substitutions of putative Site 3 residues (Y92A, E114A and H128A) are functional as repressors, but are unexpectedly compromised in their ability to sense H(2)O(2). Consistently, these mutants utilize Mn(II) but not Fe(II) as a co-repressor in vivo. Metal titrations failed to identify a third binding site in PerR, and inspection of the PerR structure suggests that these residues instead constitute a hydrogen binding network that modulates the architecture, and consequently the metal selectivity, of Site 2. PerR H128A binds DNA with high affinity, but has a significantly reduced affinity for Fe(II), and to a lesser extent for Mn(II). The ability of PerR H128A to bind Fe(II) in vivo and to thereby respond efficiently to H(2)O(2) was restored in a fur mutant strain with elevated cytosolic iron concentration.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ma
authors.forename: Jin-Won
authors.initials: JW
authors.lastname: Lee
authors.forename: John D
authors.initials: JD
authors.lastname: Helmann
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000737:chemistry / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D008670:Metals; D012097:Repressor Proteins; C122138:peroxide repressor proteins; D008345:Manganese; D004247:DNA; D006861:Hydrogen Peroxide; D007501:Iron