A novel Zn(II)/Pb(II)/Cd(II)-responsive operon that consists of genes encoding a Zn(II)/Pb(II) CPx-ATPase efflux pump (aztA) and a Zn(II)/Cd(II)/Pb(II)-specific SmtB/ArsR family repressor (aztR) has been identified and characterized from the cyanobacterium Anabaena PCC 7120. In vivo real time quantitative RT-PCR assays reveal that both aztR and aztA expression are induced by divalent metal ions Zn(II), Cd(II), and Pb(II) but not by other divalent [Co(II), Ni(II)] or monovalent metal ions [Cu(I) and Ag(I)]. The introduction of a plasmid containing the azt operon into a Zn(II)/Cd(II)-hypersensitive Escherichia coli strain GG48 functionally restores Zn(II) and Pb(II) resistance with a limited effect on Cd(II) resistance. Gel mobility shift assays and aztR O/P-lacZ induction experiments confirm that AztR is the metal-regulated repressor of this operon. In vitro biochemical and mutagenesis studies indicate that AztR contains a sole metal-binding site, designated the alpha3N site, that binds Zn(II), Cd(II), and Pb(II) with a high affinity. Optical absorption spectra of Co(II)- and Cd(II)-substituted AztR and (113)Cd NMR spectroscopy of (113)Cd(II)-substituted AztR reveal that the sole alpha3N site in AztR is a CadC-like distorted tetrahedral S(3)(N,O) metal site. The first metal-coordination shell in the AztR alpha3N site differs from other alpha3N family members that sense Cd(II)/Pb(II) and those alpha5 repressors that sense Zn(II)/Co(II). Our results reveal that the alpha3N site in AztR mediates derepression of the azt operon in the presence of Zn(II), as well as Cd(II) and Pb(II); this might have provided Anabaena with an evolutionary advantage to adapt to heavy-metal-rich environments, while maintaining homeostasis of an essential metal ion, Zn(II).
"For some proteins, negative cooperativity between the two ligand binding sites suggests that binding of a single ligand is sufficient to effect regulation. Examples include the cAMP-receptor protein (CRP) (23,24), the mercury-sensing activator MerR (25), and ArsR family metalloregulators M. tuberculosis CmtR and Anabaena AztR (26,27) and S. aureus CzrA (18). In other cases, binding of effectors to both monomers is required to elicit a regulatory response. "
[Show abstract][Hide abstract] ABSTRACT: Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.
Nucleic Acids Research 02/2009; 37(4):1174-81. DOI:10.1093/nar/gkn1052 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neurotoxic effects of inorganic lead (Pb) involve inhibition of calcium-dependent acetylcholine release and increases in calcium-dependent dopamine release. These apparently differential effects of Pb are associated with differing Pb-calcium (Ca) interactions: Pb blocks45Ca binding to peripheral cholinergic ganglia and increases45Ca binding to synaptosomes prepared from caudate nucleus (CN). Pb-induced increases in CN45Ca binding did not result from non-specific disruption of selective ion permeability of the membrane. Also, the NaK ATPase-linked Ca extrusion system of synaptosomes was not affected by Pb. A Pb-sodium (Na) interaction was found such that elevation of intrasynaptosomal Na reversed effects of Pb on45Ca binding. The intracellular localization of this effect appeared to be primarily at the mitochondrial level. Pb inhibited Na-induced release of45Ca from preloaded mitochondria. This action may be translated into increased transmembrane flux of exogenous Ca, and thence into increased exocytotic events at the synapse. The apparently neurotransmitter-specific effects of Pb, cholinergic inhibition and dopaminergic augmentation, are hypothesized to result from different PbCa interactions which are determined by the specific localization of Pb within nerve endings.
Brain Research 07/1978; 148(2-148):451-467. DOI:10.1016/0006-8993(78)90732-1 · 2.84 Impact Factor
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