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Scientific Reports Jan 2017Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although...
Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains.
Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Carboxylic Ester Hydrolases; Catalytic Domain; Escherichia coli; Molecular Conformation; Molecular Dynamics Simulation; Sequence Analysis, Protein; Streptonigrin; Substrate Specificity
PubMed: 28074848
DOI: 10.1038/srep40254 -
Antimicrobial Agents and Chemotherapy Dec 1982Mutants of Escherichia coli K-12 that are unable to make use of the enterochelin transport system were used to confirm that streptonigrin requires iron for its...
Mutants of Escherichia coli K-12 that are unable to make use of the enterochelin transport system were used to confirm that streptonigrin requires iron for its bactericidal action. Correlation of viability studies and 55Fe3+ uptake experiments showed that killing by streptonigrin increased with an increase in 55Fe3+ uptake by the cells. Streptonigrin did not kill iron-starved mutants that were unable to import iron. The level of iron uptake by these mutants was manipulated by agents such as (i) the enterochelin biosynthetic precursors 2,3-dihydroxybenzoic acid (2 x 10(-5) M) and shikimic acid (2 x 10(-4) M), (ii) citrate (10(-2) M), which promotes iron uptake by an independent pathway, and (iii) the chelating agents desferrioxamine (2 x 10(-4) M) and orthophenanthroline (10(-4) M). Addition of the precursors shikimate and dihydroxybenzoate to strain AB2847 (aroB) and dihydroxybenzoate to strain AN193 (entA), allowing these strains to make enterochelin, resulted in an increase in Fe3+ uptake and a corresponding sharp increase in killing by streptonigrin. Addition of enterochelin itself (10(-6) M) caused an even more pronounced effect. Studies on the effect of citrate in strain AN102 (fep) showed that this mutant was not killed by streptonigrin (4 x 10(-5) M), even in the presence of citrate; however, overnight growth in citrate induced Fe3+ uptake by means of the ferric citrate transport system and resulted in killing by streptonigrin. These studies showed a clear correlation between the change in levels of intracellular iron and the bactericidal effectiveness of streptonigrin.
Topics: Bacteria; Citrates; Citric Acid; Enterobactin; Escherichia coli; Iron; Iron Radioisotopes; Streptonigrin; Time Factors
PubMed: 6218780
DOI: 10.1128/AAC.22.6.961 -
Veterinary Research Mar 2021Iron is essential for most bacteria to survive, but excessive iron leads to damage by the Fenton reaction. Therefore, the concentration of intracellular free iron must...
Iron is essential for most bacteria to survive, but excessive iron leads to damage by the Fenton reaction. Therefore, the concentration of intracellular free iron must be strictly controlled in bacteria. Riemerella anatipestifer (R. anatipestifer), a Gram-negative bacterium, encodes the iron uptake system. However, the iron homeostasis mechanism remains largely unknown. In this study, it was shown that compared with the wild type R. anatipestifer CH-1, R. anatipestifer CH-1Δfur was more sensitive to streptonigrin, and this effect was alleviated when the bacteria were cultured in iron-depleted medium, suggesting that the fur mutant led to excess iron accumulation inside cells. Similarly, compared with R. anatipestifer CH-1∆recA, R. anatipestifer CH-1∆recAΔfur was more sensitive to HO-induced oxidative stress when the bacteria were grown in iron-rich medium rather than iron-depleted medium. Accordingly, it was shown that R. anatipestifer CH-1∆recAΔfur produced more intracellular ROS than R. anatipestifer CH-1∆recA in iron-rich medium. Electrophoretic mobility shift assays showed that R. anatipestifer CH-1 Fur suppressed the transcription of putative iron uptake genes through binding to their promoter regions. Finally, it was shown that compared with the wild type, R. anatipestifer CH-1Δfur was significantly attenuated in ducklings and that the colonization ability of R. anatipestifer CH-1Δfur in various tissues or organs was decreased. All these results suggested that Fur is important for iron homeostasis in R. anatipestifer and its pathogenic mechanism.
Topics: Bacterial Proteins; Iron; Oxidative Stress; Riemerella; Virulence
PubMed: 33741064
DOI: 10.1186/s13567-021-00919-9 -
Infection and Immunity Aug 2002Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene Streptococcus...
Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene Streptococcus pneumoniae operon, named pit, encoding proteins with similarity to components of a putative Brachyspira hyodysenteriae iron uptake ABC transporter, Bit. An S. pneumoniae strain containing a defined mutation in pit has impaired growth in medium containing the iron chelator ethylenediamine di-o-hydroxyphenylacetic acid, reduced sensitivity to the iron-dependent antibiotic streptonigrin, and impaired virulence in a mouse model of S. pneumoniae systemic infection. Furthermore, addition of a mutation in pit to a strain containing mutations in the two previously described S. pneumoniae iron uptake ABC transporters, piu and pia, resulted in a strain with impaired growth in two types of iron-deficient medium, a high degree of resistance to streptonigrin, and a reduced rate of iron uptake. Comparison of the susceptibilities to streptonigrin of the individual pit, piu, and pia mutant strains and comparison of the growth in iron-deficient medium and virulence of single and double mutant strains suggest that pia is the dominant iron transporter during in vitro and in vivo growth.
Topics: ATP-Binding Cassette Transporters; Bacterial Outer Membrane Proteins; Bacterial Proteins; Chlorides; Culture Media; Ferric Compounds; Gene Expression Regulation, Bacterial; Iron; Iron-Binding Proteins; Mutagenesis; Operon; Periplasmic Binding Proteins; Sequence Analysis, DNA; Streptococcus pneumoniae; Streptonigrin; Transcription, Genetic; Virulence
PubMed: 12117949
DOI: 10.1128/IAI.70.8.4389-4398.2002 -
Molecular Microbiology Dec 2008In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE),...
In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the high-pathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcus zooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbI and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.
Topics: Amino Acid Sequence; Bacterial Proteins; Chlorides; Electrophoretic Mobility Shift Assay; Escherichia coli; Ferric Compounds; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Complementation Test; Molecular Sequence Data; Multigene Family; Peptide Synthases; RNA, Bacterial; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Streptococcus equi; Streptonigrin; Substrate Specificity
PubMed: 18990191
DOI: 10.1111/j.1365-2958.2008.06481.x -
Infection and Immunity Jun 2017(group A [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and,...
(group A [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (erR-regulated etal ransporter ), a P-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448Δ deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448Δ deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.
Topics: Adenosine Triphosphatases; Animals; Bacterial Proteins; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Hydrogen Peroxide; Iron; Membrane Transport Proteins; Mice; Mice, Transgenic; Mutation; Oxidative Stress; Streptococcal Infections; Streptococcus pyogenes; Streptonigrin
PubMed: 28373352
DOI: 10.1128/IAI.00140-17 -
The Journal of Antibiotics Jun 1989Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV) and human immunodeficiency virus reverse transcriptases. The... (Comparative Study)
Comparative Study
Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV) and human immunodeficiency virus reverse transcriptases. The carboxyl group at 2'-position of STN-COOH was modified to give esters, hydrazide, amides and amino acid derivatives for biological studies. Against AMV reverse transcriptase, the hydrazide, amides and amino acid derivatives showed inhibitory activity, which compared favorably to that of STN-COOH, with the ID50 values ranging 2-8 micrograms/ml. In contrast, the esters lacked this activity except for those having a dimethylamino group in the substituent. Splenomegaly caused by Friend leukemia virus infection was significantly inhibited by STN-COOH and STN-COO(CH2)3N(CH3)2, but not STN-CONH(CH2)3N(CH3)2. Doxorubicin-resistant murine lymphoblastoma L5178Y cells showed collateral sensitivity to both STN-COOH and STN-COO(CH2)3N(CH3)2 not only in vitro but also in vivo.
Topics: Animals; Antibiotics, Antineoplastic; Antiviral Agents; Chromatography, Gel; Chromatography, Thin Layer; Circular Dichroism; Friend murine leukemia virus; Isomerism; Leukemia, Experimental; Lymphoma, Non-Hodgkin; Male; Mice; Molecular Structure; Streptonigrin; Tumor Cells, Cultured
PubMed: 2737955
DOI: 10.7164/antibiotics.42.968 -
Annals of Family Medicine 2010Findings are conflicting about the relationship between vitamin D levels and cardiovascular mortality. We wanted to determine the contribution of vitamin D levels to...
PURPOSE
Findings are conflicting about the relationship between vitamin D levels and cardiovascular mortality. We wanted to determine the contribution of vitamin D levels to black-white disparities in cardiovascular mortality.
METHODS
We examined the association of serum 25(OH)D levels with cardiovascular mortality and its contribution to elevated risk among blacks through a retrospective cohort using baseline data from the third National Health and Nutrition Examination Survey 1988-1994 and cause-specific mortality through 2001 using the National Death Index. Using piecewise Poisson regression models, we examined the risk of cardiovascular death (coronary heart disease, heart failure, and stroke) by sample 25(OH)D quartile, adjusting for cardiovascular risk factors, and compared models of adjusted race-related cardiovascular mortality with and without further adjustment for 25(OH)D levels.
RESULTS
Participants with 25(OH)D levels in the lowest quartile (mean = 13.9 ng/mL) compared with those in the 3 higher quartiles (mean = 21.6, 28.4, and 41.6 ng/mL) had higher adjusted risk of cardiovascular death (incident rate ratio [IRR] = 1.40; 95% confidence interval [CI], 1.16-1.70). The higher age- and sex-adjusted cardiovascular mortality observed in blacks vs whites (IRR = 1.38; 95% CI, 1.13-1.70) was attenuated (IRR = 1.14; 95% CI, 0.91-1.44) by adjustment for 25(OH)D levels and fully eliminated with further adjustment for income (IRR=1.01; 95% CI, 0.82-1.24).
CONCLUSIONS
Low serum levels of 25(OH)D are associated with increased cardiovascular mortality in a nationally representative US sample. Black-white differences in 25(OH)D levels may contribute to excess cardiovascular mortality in blacks. Interventional trials among persons with low vitamin D levels are needed to determine whether oral supplementation improves cardiovascular outcomes.
Topics: Adult; Black People; Cardiovascular Diseases; Cohort Studies; Female; Health Status Disparities; Humans; Hydroxycholecalciferols; Male; Middle Aged; Nutrition Surveys; Odds Ratio; Streptonigrin; United States; Vitamin D Deficiency; White People
PubMed: 20065273
DOI: 10.1370/afm.1035 -
Proceedings of the National Academy of... Dec 1986We have studied the mutagenicity (by selecting for mutants resistant to 6-thioguanine) and cytotoxicity (by determining cellular cloning efficiency) of physical and...
We have studied the mutagenicity (by selecting for mutants resistant to 6-thioguanine) and cytotoxicity (by determining cellular cloning efficiency) of physical and chemical agents in Chinese hamster ovary (CHO) cells, clone CHO-K1-BH4 (K1-BH4), and its radiation-hypersensitive transformant, AS52. AS52 cells contain a single functional copy of a bacterial gene, the xanthine/guanine phosphoribosyltransferase (gpt) gene instead of its mammalian equivalent, the hypoxanthine/guanine phosphoribosyltransferase (hprt) gene. We found that x-ray and neutron irradiations are equally toxic to both cell types; however, these physical agents are approximately equal to 10 times more mutagenic to AS52 cells than to K1-BH4 cells. Our earlier studies using Southern blot analysis showed that x-irradiation produces mostly or exclusively deletion mutations in both cell types. If reactive oxygen species mediate the mutagenic effects of radiations and chemicals, then radiomimetic compounds such as streptonigrin and bleomycin, which exert their biological effects via reactive oxygen species, and oxidizing compounds such as potassium superoxide and hydrogen peroxide should elicit a similar differential mutagenic response in both cell types. On the other hand, agents such as ethyl methanesulfonate, ICR 191, and UV light, which do not produce reactive oxygen species, should not elicit differential mutagenicity. Our results fulfill such predictions. The apparent hypermutability of AS52 cells probably results from a higher recovery of multilocus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants.
Topics: Animals; Bleomycin; Cell Line; Cell Survival; Cricetinae; DNA; DNA Damage; Free Radicals; Hydrogen Peroxide; Mutation; Neutrons; Oxygen; Streptonigrin; Superoxides; X-Rays
PubMed: 2432598
DOI: 10.1073/pnas.83.24.9616 -
Biochemical and Biophysical Research... May 2017Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to...
Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p < 0.05), whereas Pad2 levels did not change significantly (p > 0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.
Topics: Animals; Arginine; Burns, Chemical; Citrulline; Eye Burns; Female; Gliosis; Hydrolases; Male; Mice; Protein-Arginine Deiminase Type 4; Retina; Retinal Diseases; Sodium Hydroxide
PubMed: 28400047
DOI: 10.1016/j.bbrc.2017.04.031