While lanthanide-dependent metabolism is widespread in nature and has been proven to drive one-carbon metabolism in bacteria, details about the machinery necessary to sense, sequester, and traffic lanthanides (Ln) remain unknown. This gap in knowledge is in part because nearly all bacterial growth studies with Ln to date have used soluble chloride salts, compounds that do not reflect the insoluble Ln sources common in the natural environment. Here, we describe the changes in the metabolic machinery of Methylorubrum extorquens AM1 in response to poorly soluble Nd2O3, including 4-fold increases in orphan pqqA genes and the Ln-dependent ADHs xoxF1 and exaF compared to growth with soluble NdCl3. We report the first description of a Ln-chelator biosynthetic gene cluster, encoded by META1p4129 through META1p4138 that we named the Lanthanide Chelation Cluster (LCC). The LCC encodes a TonB dependent receptor and NRPS biosynthetic enzymes and is predicted to produce a metal-chelating molecule. As some LCC protein sequences share similarity to biosynthetic enzymes producing the Fe-chelating siderophore aerobactin, the capacity of aerobactin for binding Ln was tested. It was found that while aerobactin can bind lanthanum (La), neodymium (Nd) and lutetium (Lu) at physiological pH, providing only exogenous aerobactin did not affect growth rate or yield. The LCC was highly upregulated when M. extorquens AM1 was grown using Nd2O3 and expression in trans enabled an increase of Nd bioaccumulation by over 50%. Expression of the LCC in trans did not affect iron bioaccumulation, providing further evidence that its product is a novel Ln-chelator. Finally, expression of the LCC in trans increased Nd, dysprosium (Dy), and praseodymium (Pr) bioaccumulation from the complex Ln source NdFeB magnet swarf by over 60%, opening new strategies for sustainable recovery of these critical Rare Earth Elements.
Taking a closer look at Lanmodulin’s remarkable selectivity for lanthanides (Ln) over Ca(ii) and high Ln/actinide affinities on the amino acid level by investigating the four binding-loops as peptides with Ca(ii), Eu(iii), Tb(iii) and Cm(iii).
Due to the increasing demand for formaldehyde as a building block in the chemical industry as well as its emerging potential as feedstock for biofuels in the form of dimethoxymethane and the oxymethylene ethers produced therefrom, the catalytic transformation of carbon dioxide to the formaldehyde oxidation state has become a focus of interest. In this work, we present novel ruthenium complexes with hetero-triphos ligands, which show high activity in the selective transformation of carbon dioxide to dimethoxymethane. We substituted the apical carbon atom in the backbone of the triphos ligand platform with silicon or phosphorus and optimized the reaction conditions to achieve turnover numbers as high as 685 for dimethoxymethane. The catalytic systems could also be tuned to preferably yield methyl formate with turnover numbers of up to 1370, which in turn can be converted into dimethoxymethane under moderate conditions.
Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of smallmolecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation.
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Ethics approval statementAll human and animal studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
Since the discovery of the biological relevance of lanthanides (Lns) for methylotrophic bacteria in the last decade, the field has seen a steady rise in discoveries of bacteria using Lns. The major role of lanthanides here is in the active sites of enzymes: methanol dehydrogenases. Additionally, lanthanide binding proteins have also been identified. One such protein is lanmodulin (LanM) and, with a remarkable selectivity for Lns over Ca(II) and affinities in the picomolar range, it makes an attractive target to address challenges in lanthanide separation. Why LanM has such a high selectivity is currently not entirely understood, both the specific amino acid sequences of the EF-hand loops, together with cooperativity effects have been suggested. Consequently, we decided to remove the effect of cooperativity by focusing on the amino acid level. Thus, we synthesized all four 12-amino acid EF-Hand loop peptides of LanM using solid phase peptide synthesis and investigated their affinity for Lns (Eu(III), Tb(III)), the actinide Cm(III) and Ca(II). Using isothermal titration calorimetry and time resolved laser fluorescence spectroscopy combined with parallel factor analysis, we show that in the absence of cooperativity the short EF-Hand loop peptides have all similar affinities for lanthanides and that these are all in the micromolar range. Furthermore, calcium was shown not to bind to the peptides which was verified with circular dichroism spectroscopy. This technique also revealed that the peptides undergo a change to a more ordered state when lanthanides are added. These experimental observations were further supported by molecular dynamics simulations. Lastly, we put Eu(III) and Cm(III) in direct competition using TRLFS. Remarkably, a slightly higher affinity for the actinide, as was also observed for LanM, was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring in the full protein and therefore reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.
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