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C-diazeniumdiolate siderophores are a small class of photoactive bacterial Fe(III) chelators. Driven by genome mining, we discovered a new C-type diazeniumdiolate siderophore, pandorachelin, produced by the rhizospheric bacterium, Pandoraea norimbergensis DSM 11628. The biosynthetic gene cluster encoding the production of pandorachelin is conserved across several Pandoraea species. Pandoraea spp. are environmentally widespread and are increasingly prevalent clinical pathogens, spurring new interest in their metabolites. UV irradiation photolytically cleaves the N–N bonds within the diazeniumdiolate-containing graminine constituents of pandorachelin. With EPR spin trapping, we directly detect nitric oxide released from the two C-diazeniumdiolate ligands of pandorachelin upon UV irradiation. Additionally, we show that nitric oxide can react with the intermediates during the photoreaction to re-construct the diazeniumdiolate groups via exchange of the distal NO, and thereby recover Fe(III)-binding capacity. The photochemistry of this class of siderophores points to a broader biological role, both in their propensity to release the biological signaling molecule, nitric oxide, and in their ability to undergo photoinduced NO exchange. 

Yersinia pestis, the pathogen causing plague, requires iron to grow. Y. pestis employs several uptake pathways for iron, including the siderophore yersiniabactin, as well as hemin and inorganic iron. The Y. pestis iron assimilation repertoire further harbors the uncharacterized YiuRABC pathway, presumed to transport an as yet unidentified Fe(III)-siderophore(s). Through intrinsic fluorescence quenching of the periplasmic binding protein YiuA, we discovered that YiuA displays high affinity towards Fe(III) complexes of the hydrolysis products of enterobactin, Fe(III)-[di-(DHB-LSer)] and Fe(III)-[DHB-LSer]2, with Kd‘s of 27.6 ± 4.2 nM and 28.2 ± 6.9 nM, respectively, as well as the bis-catechol siderophore butanochelin, with Kd 0.76 ± 0.17 nM. By comparison, YiuA has a much weaker affinity for intact Fe(III)-enterobactin, Kd 444.7 ± 20.6 nM. Electronic circular dichroism spectroscopy reveals YiuA has a strong preference for binding Λ configured Fe(III)-siderophores, which can be achieved with the Fe(III) bis-catechol complexes but not Fe(III)-enterobactin. 

To overcome iron starvation, microorganisms often produce siderophores—chelators with high affinity and selectivity for Fe(III). The recent discovery of the siderophore gramibactin garnered significant interest, as it added the C-diazeniumdiolate as a new Fe(III)-binding group in siderophores. Gramibactin is a mixed ligand siderophore, comprised of two graminine residues harboring the diazeniumdiolate donors and a β-hydroxy-aspartate donor. Diazeniumdiolate siderophores have so far evaded crystallographic characterization and few structures of synthetic diazeniumdiolate complexes are reported. To address the gap in structural information, the complexes K[M(III)-gramibactin] (M= Fe and Ga) were prepared, crystallized and their structures solved by X-ray diffraction (XRD). The four Fe-O bond lengths in the two diazeniumdiolates are quite similar, ranging from 1.978 Å to 2.059 Å, indicating an equal contribution in bonding. In contrast, the differing Fe-O bond lengths in β-hydroxy-aspartate reflect the relative donor strengths of the carboxylate (1.997 Å) and alkoxide (1.902 Å) groups. Gramibactin coordinates Fe(III) in a Δ-configured distorted octahedral geometry. The diamagnetic nature of Ga(III) is often leveraged in NMR studies to infer the solution structure of the corresponding Fe(III)-siderophores, which are assumed to be identical. The structural similarity of Ga(III)- and Fe(III)-gramibactin is striking and represents the first crystallographic verification of the assumed isostructural relationship between a Ga(III)- and an Fe(III)-siderophore. By providing concrete evidence, this study promotes Ga(III) as a reliable proxy for Fe(III) in siderophore complexes, with implications for solution structure determination of siderophores and design of Ga(III)-siderophore-based theranostics. 

Bacteria compete for iron by producing small-molecule chelators known as siderophores. The triscatechol siderophores trivanchrobactin and ruckerbactin, produced byVibrio campbelliiDS40M4 andYersinia ruckeriYRB, respectively, are naturally occurring diastereomers that form chiral ferric complexes in opposing enantiomeric configurations. Chiral recognition is a hallmark of specificity in biological systems, yet the biological consequences of chiral coordination compounds are relatively unexplored. We demonstrate stereoselective discrimination of microbial growth and iron uptake by chiral Fe(III)–siderophores. The siderophore utilization pathway inV. campbelliiDS40M4 is stereoselective for Λ-Fe(III)–trivanchrobactin, but not the mismatched Δ-Fe(III)–ruckerbactin diastereomer. Chiral recognition is likely conferred by the stereospecificity of both the outer membrane receptor (OMR) protein FvtA and the periplasmic binding protein (PBP) FvtB, both of which must interact preferentially with the Λ-configured Fe(III)-coordination complexes. 

Yersinia ruckeriproduces the tri-catechol siderophore ruckerbactin, Rb, yet its periplasmic binding protein YiuA has unprecedented selectivity for the 1 : 2 Fe(iii) complex of the mono-catechol siderophore, Fe(iii)–(Rb_MC)₂. 

Most bacteria require iron to grow, yet soluble forms of iron are largely not available to microbes due to a combination of low solubility of ferric ion in the environment and sequestration in proteins and enzymes in living organisms. Microbes therefore compete for iron in various ways, including by production of siderophores, which are ligands with a high affinity for ferric ion and which facilitate transport of Fe(III) into and within bacteria. This review summarizes our work on the classes of siderophores isolated from open ocean isolates, including suites of amphiphilic siderophores that vary in the nature of the fatty acid appendages, photoreactive Fe(III)-siderophore complexes as a result of coordination to -hydroxy carboxylic acid groups, and a new series of tris catechol siderophores. 

Abstract Amphi-enterobactin is an amphiphilic siderophore isolated from a variety of microbialVibriospecies. Like enterobactin, amphi-enterobactin is a triscatecholate siderophore; however, it is framed on an expanded tetralactone core comprised of fourl-Ser residues, of which onel-Ser is appended by a fatty acid and the remainingl-Ser residues are appended by 2,3-dihydroxybenzoate (DHB). Fragments of amphi-enterobactin composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA have been identified in the supernatant ofVibrio campbelliispecies. The origin of these fragments has not been determined, although two distinct isomers could exist for 2-Ser-1-DHB-FA and three distinct isomers could exist for 3-Ser-2-DHB-FA. The fragments of amphi-enterobactin could originate from hydrolysis of the amphi-enterobactin macrolactone, or from premature release due to an inefficient biosynthetic pathway. Unique masses in the tandem MS analysis establish that certain fragments isolated from the culture supernatant must originate from hydrolysis of the amphi-enterobactin macrolactone, while others cannot be distinguished from premature release during biosynthesis or hydrolysis of amphi-enterobactin. Graphical abstract 

The vast majority of bacteria require iron to grow. A significant iron acquisition strategy is the production of siderophores, which are secondary microbial metabolites synthesized to sequester iron(III). Siderophore structures encompass a variety of forms, of which highly modified peptidic siderophores are of interest herein. State‐of‐the‐art genome mining tools, such as antiSMASH (antibiotics & Secondary Metabolite Analysis SHell), hold the potential to predict and discover new peptidic siderophores, including a combinatoric suite of triscatechol siderophores framed on a triserine‐ester backbone of the general class, (DHB‐ l / d CAA‐ l Ser) 3 (CAA, cationic amino acid). Siderophores with l / d Arg, l / d Lys and l Orn, but not d Orn, were predicted in bacterial genomes. Fortuitously the d Orn siderophore was identified, yet its lack of prediction highlights the limitation of current genome mining tools. The full combinatoric suite of these siderophores, which form chiral iron(III) complexes, reveals stereospecific coordination chemistry encoded in microbial genomes. The chirality embedded in this suite of Fe(III)‐siderophores raises the question of whether the relevant siderophore‐mediated iron acquisition pathways are stereospecific and selective for ferric siderophore complexes of a defined configuration. 

Ferric complexes of triscatechol siderophores may assume one of two enantiomeric configurations at the iron site. Chirality is known to be important in the iron uptake process, however an understanding of the molecular features directing stereospecific coordination remains ambiguous. Synthesis of the full suite of (DHB L/D Lys L/D Ser) 3 macrolactone diastereomers, which includes the siderophore cyclic trichrysobactin (CTC), enables the effects that the chirality of Lys and Ser residues exert on the configuration of the Fe( iii ) complex to be defined. Computationally optimized geometries indicate that the Λ/Δ configurational preferences are set by steric interactions between the Lys sidechains and the peptide backbone. The ability of each (DHB L/D Lys L/D Ser) 3 diastereomer to form a stable Fe( iii ) complex prompted a genomic search for biosynthetic gene clusters (BGCs) encoding the synthesis of these diastereomers in microbes. The genome of the plant pathogen Dickeya chrysanthemi EC16 was sequenced and the genes responsible for the biosynthesis of CTC were identified. A related but distinct BGC was identified in the genome of the opportunistic pathogen Yersinia frederiksenii ATCC 33641; isolation of the siderophore from Y. frederiksenii ATCC 33641, named frederiksenibactin (FSB), revealed the triscatechol oligoester, linear -(DHB L Lys L Ser) 3 . Circular dichroism (CD) spectroscopy establishes that Fe( iii )–CTC and Fe( iii )–FSB are formed in opposite enantiomeric configuration, consistent with the results of the ferric complexes of the cyclic (DHB L/D Lys L/D Ser) 3 diastereomers.