Siderophore Pathways Research Articles

Feature sequence-based genome mining uncovers the hidden diversity of bacterial siderophore pathways.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

Chemoenzymatic Synthesis of Select Intermediates and Natural Products of the Desferrioxamine E Siderophore Pathway.

The NIS synthetase family of enzymes responsible for the biosynthesis of siderophores is increasingly associated with bacterial virulence. Proteins in this class represent outstanding potential drug targets, assuming that basic biochemical and structural characterizations can be completed. Towards this goal, we have mated an improved synthesis of the non-commercial amino acid N-hydroxy-N-succinylcadaverine (HSC, 6) with an isothermal titration calorimetry (ITC) assay that profiles the iterative stages of HSC trimerization and macrocyclization by NIS synthetase DesD from Streptomyces coelicolor. HSC synthesis begins with multigram-scale Gabrielle and tert-butyl N-(benzyloxy)carbamate alkylations of 1-bromo-5-chloropentane following prior literature, but the end-game reported herein has two advantages for greater material throughput: (1) hydrogenolysis of benzyl ether and Cbz blocking groups is best accomplished with Pearlman’s catalyst at 40 psi of H2 and (2) purification of neutral (zwitterionic) HSC is effected by simple flash chromatography over silica gel in MeOH. HSC is subsequently shown to be a substrate for NIS synthetase DesD, which catalyzes three successive amide bond syntheses via adenyl monophosphate ester intermediates. We quantify and present the iterative and overall enzyme kinetic constants associated with formation of the cyclotrimeric siderophore desferrioxamine E (dfoE, 1).

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa.

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, 55 Fe uptake assays, and LC-MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.

Isolation and Characterization of Strain Exiguobacterium sp. KRL4, a Producer of Bioactive Secondary Metabolites from a Tibetan Glacier.

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 °C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds.

Determining the Kinetics of Desferroxiamine D from Streptomyces coelicolor using Isothermal Titration Calorimetry

The long‐term objective for this research is to create a new class of antibiotics that targets the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway in bacteria. The focus of my current research is to quantify the first and second order kinetic constants and obtain the kinetics curve of our model system, Desferroxiamine D (DesD), from Streptomyces coelicolor. DesD reacts with ATP as a cofactor, and either the small (hydroxysuccinylcadaverine, HSC), medium or large (desferrioxamine G, or dfoG) substrates in an iterative manner. We have so far collected activity curves with ATP and dfoG limiting, and are pursuing HSC curves. To do this we used Isothermal Titration Calorimetry (ITC) to collect data and GraphPad Prism 8.2 along with Malvern MicroCal Peaq ITC software to fit the kinetics curves and determine the kinetics constants. We successfully gathered and analyzed ITC data for both the catalytic (wild type, Wt) and non‐catalytic (mutant, R306Q) forms of DesD and determined the optimal buffer conditions for solubility. We also gathered data regarding the activity of our Wt DesD enzyme in order to determine the lifespan and the time of optimal catalytic activity. We have found that our enzyme is still active within 96 hours where it was previously thought to have a 36–48 hour catalytic lifespan.In conducting these experiments, we observed that an industry standard amount of magnesium chloride (MgCl2) in our solution caused an oscillating signal during equilibration of the ITC prior to enzyme injection. We hypothesized that the data reflects metal chelation of the Mg by our substrate (dfoG). We plan on conducting a binding experiment using the ITC in order to quantify the substrate (dfoG) and MgCl2 interaction, and ultimately recommend a new standard amount of MgCl2 to the field for future experiments. Other future directions include determining the kinetic constants of Wt DesD interacting with its small substrate HSC (which must be synthesized and purified,) and determining the catalytic role of other residues in the active site by site‐directed mutagenesis and ITC. This experimental assay and data will be the foundation for future drug design targeting DesD.Support or Funding InformationThis research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.

Large and Small Substrate Binding to DesD via ITC Assay Development

Desferrioxamines (“dfo”s) are iron‐chelating compounds which have been used medicinally in cases of iron overload. The synthesis of dfoE involves an enzyme called DesD which is a dimer that has an active site on each chain, but the mechanism of it binding both large or small substrates is unknown. We are studying DesD as a model enzyme for the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway and as a potential new drug target. The purpose of this research is to analyze the co‐factor specificity and large substrate verse small substrate specificity of DesD by comparing substrate dissociation constants to analog dissociation constants (KD, where a smaller number correlates to tighter binding). Future drugs will have to out‐compete the substrates in binding, so we will use these studies to determine which substrate components contribute to maximum binding. Techniques in analytical chemistry will be used to regenerate deferoxamines that were bound to cofactors in previous studies with the DesD enzyme. The binding properties of DesD were explored in a 25% glycerol Isothermal Titration Calorimetry (ITC) buffer. Prior experimentation with this protein was conducted in a buffer that resulted in precipitation of DesD out of solution, thus prompting the need for repeated binding assays in a buffer that maintains synthase solubility. GraphPad Prism 8.0 will be used to analyze the KD and occupancy of DesD. High Performance Liquid Chromatography (HPLC) will be used to separate deferoxamines from substrates they were bound to. An Ocean Optics Spectrometer will be used to quantify the deferoxamines by pairing these substrates to a metal compound that will allow us to identify its absorbance and determine if isolation was effective.We confirmed previous results that the KD of DesD binding to ATP is in the M range, compared to non‐ATP cofactors which result in a KD in the M range. Future studies will compare dfoG (substrate) binding to DesD compared to analog dfoB or product dfoE, and also explore smaller substrates, hydroxysuccinylcadaverine (HSC), vs. analogs. Troubleshooting runs have indicated that isolation of the deferoxamines is feasible. It is expected that substrates like dfoG will be separated from ATP and ADP cofactors. We have confirmed that DesD has a strong binding preference for ATP over non‐ATP cofactors in a buffer that maximizes solubility. This data indicates that the phosphates are essential to tight binding in any future competitive inhibitors. Regeneration of each deferoxamine appeared to be a cost‐effective assay that will allow for these compounds to be used in later experimentation. Future work will expand our understanding of critical regions in the binding site, and aid in developing new inhibitors that could block the NIS pathways and be lead drugs for a new class of antibiotics.

Flocking asbestos waste, an iron and magnesium source for Pseudomonas

Iron and magnesium are essential nutrients for most microorganisms. In the environment, the availability of iron is low relative to that of magnesium. Microorganisms have developed various iron acquisition systems, which have been well studied, whereas few studies have examined magnesium acquisition. The production of siderophores is one of the efficient strategies widely used to sustain iron nutritional requirements. Many studies have shown that minerals, such as clays, iron oxides, and silicates, can serve as nutrient sources for bacteria. Asbestos, a natural fibrous silicate present in soil contains iron and/or magnesium, depending on the species of asbestos. Our aim was to study the acquisition of iron and magnesium from flocking asbestos waste by Pseudomonas aeruginosa and the involvement of the siderophores, pyoverdine and pyochelin. Flocking asbestos waste promoted growth under iron- and magnesium-limited conditions, together with a decrease in pyoverdine production, correlating with the dissolution of iron from the waste. In long-term experiments, flocking asbestos waste provided these two essential elements for bacterial growth and resulted in a decrease of iron in asbestos fibers. Among the enzymes required for pyochelin and pyoverdine synthesis, PchA and PvdJ were tagged with the fluorescent protein mCherry to analyze the expression patterns of proteins involved in siderophore production. Both enzymes were produced in the presence of flocking asbestos waste, suggesting a role of the pyoverdine and pyochelin pathway in asbestos dissolution. We investigated the involvement of each siderophore in iron and magnesium removal using mutants in one or both siderophore pathways. We observed a significant increase in iron extraction in the presence of siderophores and the absence of one of the two siderophores could be compensated by the other. Flocking asbestos waste represents an iron and magnesium source for P. aeruginosa, with iron removal linked to a siderophore-driven mechanism.

Ironing out pyoverdine's chromophore structure: serendipity or design?

Pyoverdines are Pseudomonas aeruginosa's primary siderophores. These molecules, composed of a fluorescent chromophore attached to a peptide chain of 6-14 amino acids, are synthesized by the bacterium to scavenge iron (essential to its survival and growth) from its environment. Hijacking the siderophore pathway to use pyoverdine-antibiotic compounds in a Trojan horse approach has shown promise but remains very challenging because of the synthetic efforts involved. Indeed, both possible approaches (grafting an antibiotic on pyoverdine harvested from Pseudomonas or designing a total synthesis route) are costly, time-consuming and low-yield tasks. Designing comparatively simple analogs featuring the salient properties of the original siderophore is thus crucial for the conception of novel antibiotics to fight bacterial resistance. In this work, we focus on the replacement of the pyoverdine chromophore, a major roadblock on the synthetic pathway. We propose three simpler analogs and evaluate their ability to complex iron and interact with the FpvA transporter using molecular modeling techniques. Based on these results, we discuss the role of the native chromophore's main features (polycyclicity, positive charge, flexibility) on pyoverdine's ability to bind iron and be recognized by membrane transporter FpvA and propose guidelines for the design of effective synthetic siderophores.

Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility

Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.

Kinetics and Binding Studies of the (NRPS)‐Independent Siderophore (NIS) Synthetase DesD

Pathogenic bacteria have always been a problem in human health. They do not discriminate when it comes to finding a human host for them to survive in, so it effects all people regardless of ethnicity and socioeconomic background. Pathogenic bacteria use their host to supply them with something that they lack. In the case of iron, bacteria synthesize, secrete, and reacquire small molecule chelators called siderophores. Our lab is specifically focusing on siderophores made with the non‐ribosomal peptide synthetase independent siderophore (NIS) pathways, which always contain at least one NIS synthetase family protein. These proteins are excellent drug targets because of their association with virulence, their unique chemistry, and their unique fold. A drug that successfully targeted and inhibited this family of proteins might represent a new class of antibiotics.The best studied NIS synthetase is Desferrioxamine synthetase D (DesD) from Streptomyces coelicolor, which makes the group of siderophores called desferrioxamines. Desferrioxamine E is the biggest siderophore made of 3 N‐hydoxysuccinylcadaverine units repeatedly added by DesD and then cyclized. This broad substrate specificity (the variation in size recognized by the same protein), is the reason why we are interested in mapping the thermodynamics of binding to different substrates to see if there is a variation in association constants. We are using Isothermal Titration Calorimetry (ITC) because it is label‐free and have successfully piloted the conditions for the assay. If we are able to delineate the priority requirements for binding, we will progress to designing an inhibitor to maximize the binding affinity. ITC methods will also be used to characterize the kinetics DesD so that a better overall picture of the proteins' biochemistry may be understood.Support or Funding InformationThis work was supported by the National Science Foundation grant to K.M.H. (MSP ‐ 1716986) and by California Lutheran University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hybrid genome assembly and annotation of Paenibacillus pasadenensis strain R16 reveals insights on endophytic life style and antifungal activity.

Bacteria of the Paenibacillus genus are becoming important in many fields of science, including agriculture, for their positive effects on the health of plants. However, there are little information available on this genus compared to other bacteria (such as Bacillus or Pseudomonas), especially when considering genomic information. Sequencing the genomes of plant-beneficial bacteria is a crucial step to identify the genetic elements underlying the adaptation to life inside a plant host and, in particular, which of these features determine the differences between a helpful microorganism and a pathogenic one. In this study, we have characterized the genome of Paenibacillus pasadenensis, strain R16, recently investigated for its antifungal activities and plant-associated features. An hybrid assembly approach was used integrating the very precise reads obtained by Illumina technology and long fragments acquired with Oxford Nanopore Technology (ONT) sequencing. De novo genome assembly based solely on Illumina reads generated a relatively fragmented assembly of 5.72 Mbp in 99 ungapped sequences with an N50 length of 544 Kbp; hybrid assembly, integrating Illumina and ONT reads, improved the assembly quality, generating a genome of 5.75 Mbp, organized in 6 contigs with an N50 length of 3.4 Mbp. Annotation of the latter genome identified 4987 coding sequences, of which 1610 are hypothetical proteins. Enrichment analysis identified pathways of particular interest for the endophyte biology, including the chitin-utilization pathway and the incomplete siderophore pathway which hints at siderophore parasitism. In addition the analysis led to the identification of genes for the production of terpenes, as for example farnesol, that was hypothesized as the main antifungal molecule produced by the strain. The functional analysis on the genome confirmed several plant-associated, plant-growth promotion, and biocontrol traits of strain R16, thus adding insights in the genetic bases of these complex features, and of the Paenibacillus genus in general.

An Engineered Synthetic Pathway for Discovering Nonnatural Nonribosomal Peptides in Escherichiacoli.

ABSTRACTPeptides that are synthesized independently of the ribosome in plants, fungi, and bacteria can have clinically relevant anticancer, antihemochromatosis, and antiviral activities, among many other. Despite their natural origin, discovering new natural products is challenging, and there is a need to expand the chemical diversity that is accessible. In this work, we created a novel, compressed synthetic pathway for the heterologous expression and diversification of nonribosomal peptides (NRPs) based on homologs of siderophore pathways from Escherichia coli and Vibrio cholerae. To enhance the likelihood of successful molecule production, we established a selective pressure via the iron-chelating properties of siderophores. By supplementing cells containing our synthetic pathway with different precursors that are incorporated into the pathway independently of NRP enzymes, we generated over 20 predesigned, novel, and structurally diverse NRPs. This engineering approach, where phylogenetically related genes from different organisms are integrated and supplemented with novel precursors, should enable heterologous expression and molecular diversification of NRPs.

Function-related replacement of bacterial siderophore pathways

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co-occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

Microbial siderophore-based iron assimilation and therapeutic applications.

Siderophores are structurally diverse, complex natural products that bind metals with extraordinary specificity and affinity. The acquisition of iron is critical for the survival and virulence of many pathogenic microbes and diverse strategies have evolved to synthesize, import and utilize iron. There has been a substantial increase of known siderophore scaffolds isolated and characterized in the past decade and the corresponding biosynthetic gene clusters have provided insight into the varied pathways involved in siderophore biosynthesis, delivery and utilization. Additionally, therapeutic applications of siderophores and related compounds are actively being developed. The study of biosynthetic pathways to natural siderophores augments the understanding of the complex mechanisms of bacterial iron acquisition, and enables a complimentary approach to address virulence through the interruption of siderophore biosynthesis or utilization by targeting the key enzymes to the siderophore pathways.

Determining the Structure of NRPS‐Independent Siderophore (NIS) Synthetase DesD Using X‐ray Crystallography

A lack of iron leads to secretion of siderophores (small molecule metal chelators) by bacteria. Siderophores have high affinity for iron outcompeting host chelators, and making them virulence factors in infection. Some siderophores (of particularly virulent pathogens) are assembled via an NRPS‐independent siderophore (NIS) pathway. Understanding the structure of NIS siderophore biosynthesis enzymes may lead to the design of specific antibiotics to fight pathogenic bacteria.The Hoffmann lab is using x‐ray crystallography to determine the protein structure of NIS synthetase DesD, from Streptomyces coelicolor. DesD makes the siderophore desferrioxamine B, requiring three iterative bonds and a macrocyclization event. DesD is one of the best biochemically characterized NIS synthetases so far, and we are solving this structure as a model for the rest. The goal of this project is to solve the x‐ray structure of DesD bound to substrate and ATP, as well as apo (empty of substrate) structures, in order to characterize the conformational changes that may occur with binding. DesD crystalizes in 20% PED 8,000, 15% glycerol, and 15% ethylene glycol conditions, and crystals diffract to 2.48 Å with good statistics. We are solving the structure using molecular replacement and are pursuing substrate‐bound crystals. This structural information may allow us to design an inhibitor that will prevent the binding of substrate in future work.

Structure and functional analysis of the siderophore periplasmic binding protein from the fuscachelin gene cluster of Thermobifida fusca.

Iron acquisition is a complex, multicomponent process critical for most organisms' survival and virulence. Small iron chelating molecules, siderophores, mediate transport as key components of common pathways for iron assimilation in many microorganisms. The chemistry and biology of the extraordinary tight and specific metal binding siderophores is of general interest in terms of host/guest chemistry and is a potential target toward the development of therapeutic treatments for microbial virulence. The siderophore pathway of the moderate thermophile, Thermobifida fusca, is an excellent model system to study the process in Gram-positive bacteria. Here we describe the structure and characterization of the siderophore periplasmic binding protein, FscJ from the fuscachelin gene cluster of T. fusca. The structure shows a di-domain arrangement connected with a long α-helix hinge. Several X-ray structures detail ligand-free conformational changes at different pH values, illustrating complex interdomain flexibility of the siderophore receptors. We demonstrated that FscJ has a unique recognition mechanism and details the binding interaction with ferric-fuscachelin A through ITC and docking analysis. The presented work provides a structural basis for the complex molecular mechanisms of siderophore recognition and transportation.

Structure and Mechanism of the Siderophore-Interacting Protein from the Fuscachelin Gene Cluster of Thermobifida fusca

Microbial iron acquisition is a complex process and frequently a key and necessary step for survival. Among the several paths for iron assimilation, small molecule siderophore-mediated transport is a commonly employed strategy of many microorganisms. The chemistry and biology of the extraordinary tight and specific binding of siderophores to metal is also exploited in therapeutic treatments for microbial virulence and metal toxicity. The intracellular fate of iron acquired via the siderophore pathway is one of the least understood steps in the complex process at the molecular level. A common route to cellular incorporation is the single-electron reduction of ferric to ferrous iron catalyzed by specific and/or nonspecific reducing agents. The biosynthetic gene clusters for siderophores often contain representatives of one or two families of redox-active enzymes: the flavin-containing "siderophore-interacting protein" and iron-sulfur ferric siderophore reductases. Here we present the structure and characterization of the siderophore-interacting protein, FscN, from the fuscachelin siderophore gene cluster of Thermobifida fusca. The structure shows a flavoreductase fold with a noncovalently bound FAD cofactor along with an unexpected metal bound adjacent to the flavin site. We demonstrated that FscN is redox-active and measured the binding and reduction of ferric fuscachelin. This work provides a structural basis for the activity of a siderophore-interacting protein and further insight into the complex and important process of iron acquisition and utilization.

A novel antimycobacterial compound acts as an intracellular iron chelator.

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound.