Arabidopsis plants exposed to the antibiotic kanamycin (Kan) display altered metal homeostasis. Further, mutation of the WBC19 gene leads to increased sensitivity to kanamycin and changes in iron (Fe) and zinc (Zn) uptake. Here we propose a model that explain this surprising relationship between metal uptake and exposure to Kan. We first use knowledge about the metal uptake phenomenon to devise a transport and interaction diagram on which we base the construction of a dynamic compartment model. The model has three pathways for loading Fe and its chelators into the xylem. One pathway, involving an unknown transporter, loads Fe as a chelate with citrate (Ci) into the xylem. This transport step can be significantly inhibited by Kan. In parallel, FRD3 transports Ci into the xylem where it can chelate with free Fe. A third critical pathway involves WBC19, which transports metal-nicotianamine (NA), mainly as Fe-NA chelate, and possibly NA itself. To permit quantitative exploration and analysis, we use experimental time series data to parameterize this explanatory and predictive model. Its numerical analysis allows us to predict responses by a double mutant and explain the observed differences between data from wildtype, mutants and Kan inhibition experiments. Importantly, the model provides novel insights into metal homeostasis by permitting the reverse-engineering of mechanistic strategies with which the plant counteracts the effects of mutations and of the inhibition of iron transport by kanamycin.
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Aminopolycarboxylates by the Periplasmic EDTA- Binding Protein EppA from Chelativorans sp. BNC1
The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal–chelate complexes. Microorganisms can transport free EDTA, but not metal–EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure–function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal–chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.
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- Award ID(s):
- 1804699
- NSF-PAR ID:
- 10157186
- Date Published:
- Journal Name:
- International journal of molecular sciences
- Volume:
- 21
- ISSN:
- 1661-6596
- Page Range / eLocation ID:
- 3940
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Ethylenediaminetetraacetate (EDTA) is the most abundant organic pollutant in surface water because of its extensive usage and the recalcitrance of stable metal‐EDTA complexes. A few bacteria including
Chelativorans sp. BNC1 can degrade EDTA with a monooxygenase to ethylenediaminediacetate (EDDA) and then use iminodiacetate oxidase (IdaA) to further degrade EDDA into ethylenediamine in a two‐step oxidation. To alleviate EDTA pollution into the environment, deciphering the mechanisms of the metabolizing enzymes is an imperative prerequisite for informed EDTA bioremediation. Although IdaA cannot oxidize glycine, the crystal structure of IdaA shows its tertiary and quaternary structures similar to those of glycine oxidases. All confirmed substrates, EDDA, ethylenediaminemonoacetate, iminodiacetate and sarcosine are secondary amines with at least one N‐acetyl group. Each substrate was bound at there ‐side face of the isoalloxazine ring in a solvent‐connected cavity. The carboxyl group of the substrate was bound by Arg265and Arg307. The catalytic residue, Tyr250, is under the hydrogen bond network to facilitate its deprotonation acting as a general base, removing an acetate group of secondary amines as glyoxylate. Thus, IdaA is a secondary amine oxidase, and our findings improve understanding of molecular mechanism involved in the bioremediation of EDTA and the metabolism of secondary amines. -
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Abstract Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection ofS. aureus is crucial to prevent infections and ensure food quality. The iron‐regulated surface determinant protein A (IsdA) ofS. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in‐silico approach to develop and validate high‐affinity binding aptamers for the IsdA protein detection using custom‐designed in‐silico tools and single‐molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in‐silico oligonucleotide screening methods and metadynamics‐based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA‐aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in‐silico approach to support aptamer discovery.This study showcases a computational SELEX method in combination with single‐molecule binding studies deciphering effective aptamers against
S. aureus IsdA, protein. The established approach demonstrates the ability to expedite aptamer discovery that has the potential to cut costs and predict binding efficacy. The application can be extended to designing aptamers for various protein targets, enhancing molecular recognition, and facilitating the development of high‐affinity aptamers for multiple uses. -
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Abstract The central role of iron in tumor progression and metastasis motivates the development of iron‐binding approaches in cancer chemotherapy. Disulfide‐based prochelators are reductively activated upon cellular uptake to liberate thiol chelators responsible for iron sequestration. Herein, a trimethyl thiosemicarbazone moiety and the imidazole‐2‐thione heterocycle are incorporated in this prochelator design. Iron binding of the corresponding tridentate chelators leads to the stabilization of a low‐spin ferric center in 2 : 1 ligand‐to‐metal complexes. Native mass spectrometry experiments show that the prochelators form stable disulfide conjugates with bovine serum albumin, thus affording novel bioconjugate prochelator systems. Antiproliferative activities at sub‐micromolar levels are recorded in a panel of breast, ovarian and colorectal cancer cells, along with significantly lower activity in normal fibroblasts.
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Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of
Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+and Mn2+are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+is different and more robust to perturbation than for transporting the toxic Cd2+metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/ijms21113940
