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Elucidation of the functional metal binding profile of a Cd(II)/Pb(II) sensor CmtR(Sc) from Streptomyces coelicolor.

ABSTRACT: Metal homeostasis and resistance in bacteria is maintained by a panel of metal-sensing transcriptional regulators that collectively control transition metal availability and mediate resistance to heavy metal xenobiotics, including As(III), Cd(II), Pb(II), and Hg(II). The ArsR family constitutes a superfamily of metal sensors that appear to conform to the same winged helical, homodimeric fold, that collectively "sense" a wide array of beneficial metal ions and heavy metal pollutants. The genomes of many actinomycetes, including the soil dwelling bacterium Streptomyces coelicolor and the human pathogen Mycobacterium tuberculosis, encode over ten ArsR family regulators, most of unknown function. Here, we present the characterization of a homologue of M. tuberculosis CmtR (CmtR(Mtb)) from S. coelicolor, denoted CmtR(Sc). We show that CmtR(Sc), in contrast to CmtR(Mtb), binds two monomer mol equivalents of Pb(II) or Cd(II) to form two pairs of sulfur-rich coordination complexes per dimer. Metal site 1 conforms exactly to the alpha4C site previously characterized in CmtR(Mtb) while metal site 2 is coordinated by a C-terminal vicinal thiolate pair, Cys110 and Cys111. Biological assays reveal that only Cd(II) and, to a lesser extent, Pb(II) mediate transcriptional derepression in the heterologous host Mycobacterium smegmatis in a way that requires metal site 1. In contrast, mutagenesis of metal site 2 ligands Cys110 or Cys111 significantly reduces Cd(II) responsiveness, with no detectable effect on Pb(II) sensing. The implications of these findings on the ability to predict metal specificity and function from metal-site signatures in the primary structure of ArsR family proteins are discussed.

SUBMITTER: Wang Y

PROVIDER: S-EPMC2925124 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Elucidation of the functional metal binding profile of a Cd(II)/Pb(II) sensor CmtR(Sc) from Streptomyces coelicolor.

Wang Yun Y Kendall John J Cavet Jennifer S JS Giedroc David P DP

Biochemistry 20100801 31

Metal homeostasis and resistance in bacteria is maintained by a panel of metal-sensing transcriptional regulators that collectively control transition metal availability and mediate resistance to heavy metal xenobiotics, including As(III), Cd(II), Pb(II), and Hg(II). The ArsR family constitutes a superfamily of metal sensors that appear to conform to the same winged helical, homodimeric fold, that collectively "sense" a wide array of beneficial metal ions and heavy metal pollutants. The genomes ...[more]

PMID: 20586430

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Project description:BackgroundThe natural polyether ionophorous antibiotics are used for the treatment of coccidiosis in poultry and ruminants. They are effective agents against infections caused by Gram-positive microorganisms. On the other hand, it was found that some of these compounds selectively bind lead(II) ions in in vivo experiments, despite so far no Pb(II)-containing compounds of defined composition have been isolated and characterized. To assess the potential of polyether ionophores as possible antidotes in the agriculture, a detailed study on their in vitro complexation with toxic metal ions is required. In the present paper we report for the first time the preparation and the structure elucidation of salinomycin complexes with ions of cadmium(II) and lead(II).ResultsNew metal(II) complexes of the polyether ionophorous antibiotic salinomycin with Cd(II) and Pb(II) ions were prepared and structurally characterized by IR, FAB-MS and NMR techniques. The spectroscopic information and elemental analysis data reveal that sodium salinomycin (SalNa) undergoes a reaction with heavy metal(II) ions to form [Cd(Sal)2(H2O)2] (1) and [Pb(Sal)(NO3)] (2), respectively. Abstraction of sodium ions from the cavity of the antibiotic is occurring during the complexation reaction. Salinomycin coordinates with cadmium(II) ions as a bidentate monoanionic ligand through the deprotonated carboxylic moiety and one of the hydroxyl groups to yield 1. Two salinomycin anions occupy the equatorial plane of the Cd(II) center, while two water molecules take the axial positions of the inner coordination sphere of the metal(II) cation. Complex 2 consists of monoanionic salinomycin acting in polydentate coordination mode in a molar ratio of 1: 1 to the metal ion with one nitrate ion for charge compensation.ConclusionThe formation of the salinomycin heavy metal(II) complexes indicates a possible antidote activity of the ligand in case of chronic/acute intoxications likely to occur in the stock farming.

Project description:The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/mol of protein and utilizes guanosine 5'-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912-36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting a role for Tyr120 in the late phase of the reaction. Our data are consistent with duplication of GCH II in S. coelicolor promoting evolution of a new function. The physiological role(s) of the gene clusters that house GCH II homologues will be discussed.

Dataset Information

Elucidation of the functional metal binding profile of a Cd(II)/Pb(II) sensor CmtR(Sc) from Streptomyces coelicolor.

Publications

Elucidation of the functional metal binding profile of a Cd(II)/Pb(II) sensor CmtR(Sc) from Streptomyces coelicolor.

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