Enterobactin protonation and iron release: structural characterization of the salicylate coordination shift in ferric enterobactin

The siderophore enterobactin (Ent) is produced by many species of enteric bacteria to mediate iron uptake. This iron scavenger can be reincorporated by the bacteria as the ferric complex [Fe(III)(Ent)](3)(-) and is subsequently hydrolyzed by an esterase to facilitate intracellular iron release. Recent literature reports on altered protein recognition and binding of modified enterobactin increase the significance of understanding the structural features and solution chemistry of ferric enterobactin. The structure of the neutral protonated ferric enterobactin complex [Fe(III)(H(3)Ent)](0) has been the source of some controversy and confusion in the literature. To demonstrate the proposed change of coordination from the tris-catecholate [Fe(III)(Ent)](3)(-) to the tris-salicylate [Fe(III)(H(3)Ent)](0) upon protonation, the coordination chemistry of two new model compounds N,N',N'-tris[2-(hydroxybenzoyl)carbonyl]cyclotriseryl trilactone (SERSAM) and N,N',N'-tris[2-hydroxy,3-methoxy(benzoyl)carbonyl]cyclotriseryl trilactone (SER(3M)SAM) was examined in solution and solid state. Both SERSAM and SER(3M)SAM form tris-salicylate ferric complexes with spectroscopic and solution thermodynamic properties (with log beta(110)() values of 39 and 38 respectively) similar to those of [Fe(III)(H(3)Ent)](0). The fits of EXAFS spectra of the model ferric complexes and the two forms of ferric enterobactin provided bond distances and disorder factors in the metal coordination sphere for both coordination modes. The protonated [Fe(III)(H(3)Ent)](0) complex (d(Fe)(-)(O) = 1.98 A, sigma(2)(stat)(O) = 0.00351(10) A(2)) exhibits a shorter average Fe-O bond length but a much higher static Debye-Waller factor for the first oxygen shell than the catecholate [Fe(III)(Ent)](3)(-) complex (d(Fe)(-)(O) = 2.00 A, sigma(2)(stat)(O) = 0.00067(14) A(2)). (1)H NMR spectroscopy was used to monitor the amide bond rotation between the catecholate and salicylate geometries using the gallic complexes of enterobactin: [Ga(III)(Ent)](3)(-) and [Ga(III)(H(3)Ent)](0). The ferric salicylate complexes display quasi-reversible reduction potentials from -89 to -551 mV (relative to the normal hydrogen electrode NHE) which supports the feasibility of a low pH iron release mechanism facilitated by biological reductants.     

The siderocalin/enterobactin interaction: a link between mammalian immunity and bacterial iron transport

The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [Fe (III)(Ent)] (3-). This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an antibacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidic endosomes and [Fe (III)(Ent)] (3-) is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[Fe (III)(Ent)] (3-) and Scn-Y106F:[Fe (III)(Ent)] (3-) complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe (III)(Ent)] (3-). Fluorescence, UV-vis, and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogues of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.     

Facile and Versatile Chemoenzymatic Synthesis of Enterobactin Analogues and Applications in Bacterial Detection

Siderophores, such as enterobactin (Ent), are small molecules that can be selectively imported into bacteria along with iron by cognate transporters. Siderophore conjugates are thus a promising strategy for delivering functional reagents into bacteria. In this work, we present an easy‐to‐perform, one‐pot chemoenzymatic synthesis of functionalized monoglucosylated enterobactin (MGE). When functionalized MGE is conjugated to a rhodamine fluorophore, which affords RhB‐Glc‐Ent, it can selectively label Gram‐negative bacteria that utilize Ent, including some E. coli strains and P. aeruginosa. V. cholerae, a bacterium that utilizes linearized Ent, can also be weakly targeted. Moreover, the targeting is effective under iron‐limiting but not iron‐rich conditions. Our results suggest that the RhB‐Glc‐Ent probe is sensitive not only to the bacterial strain but also to the iron condition in the environment.     

Facile synthesis of salmochelin S1, S2, MGE, DGE, and TGE

Salmochelin S1, S2, MGE, DGE, and TGE were prepared through amide bond connection of an aryl C-glucosyl acyl chloride (Ar¹COCl) and serine ester amines, followed by hydrogenolysis of the per-benzylated precursors. Each synthesis employed a highly diastereoselective Ni-catalyzed Negishi approach to the aryl C-glycoside subunit.     

Bacillibactin-mediated iron transport in Bacillus subtilis

The hexadentate triscatecholamide bacillibactin delivers iron to Bacillus subtilis and is structurally similar to enterobactin, although in a more oblate conformation. B. subtilis uses two partially overlapping permeases (1 and 2) to acquire iron from its endogenous siderophores (bacillibactin and itoic acid). Enterobactin and bacillibactin have opposite metal chiralities, different affinity for ferric ion, and dissimilar iron transport behaviors. The solution thermodynamic stability of ferric bacillibactin has been investigated through potentiometric and spectrophotometric titrations. The addition of a glycine to the catechol chelating arms causes a destabilization of the ferric complex of bacillibactin compared to ferric enterobactin. B. subtilis appears to express a separate receptor for enterobactin (permease 3), although enterobactin can also be transported through the permease for bacillibactin (permease 2).     

Tren-based analogues of bacillibactin: structure and stability

Synthetic analogues were designed to highlight the effect of the glycine moiety of bacillibactin on the overall stability of the ferric complex as compared to synthetic analogues of enterobactin. Insertion of a variety of amino acids to catecholamide analogues based on a Tren (tris(2-aminoethyl)amine) backbone increased the overall acidity of the ligands, causing an enhancement of the stability of the resulting ferric complex as compared to TRENCAM. Solution thermodynamic behavior of these siderophores and their synthetic analogues was investigated through potentiometric and spectrophotometric titrations. X-ray crystallography, circular dichroism, and molecular modeling were used to determine the chirality and geometry of the ferric complexes of bacillibactin and its analogues. In contrast to the Tren scaffold, addition of a glycine to the catechol chelating arms causes an inversion of the trilactone backbone, resulting in opposite chiralities of the two siderophores and a destabilization of the ferric complex of bacillibactin compared to ferric enterobactin.     

Corynebactin and enterobactin: related siderophores of opposite chirality

Most species of bacteria employ siderophores to acquire iron. The chirality of the ferric siderophore complex plays an important role in cell recognition, uptake, and utilization. Corynebactin, isolated from Gram-positive bacteria, is structurally similar to enterobactin, a well known siderophore isolated from Gram-negative bacteria, but contains L-theronine instead of L-serine in the trilactone backbone. Corynebactin also contains a glycine spacer unit in each of the chelating arms. A hybrid analogue (serine-corynebactin) has been synthesized. The chirality and relative conformational stability of the three ferric complexes of enterobactin, corynebactin, and the hybrid has been investigated. In contrast to enterobactin, corynebactin assumes a Lambda configuration. However, the ferric serine-corynebactin hybrid forms a racemic mixture, only slightly favoring the Lambda conformation.     

Catecholate/salicylate heteropodands: demonstration of a catecholate to salicylate coordination change

While iron release from enterobactin-mediated iron transport occurs primarily via an esterase that destroys the siderophore, other catechol siderophores that are not susceptible to hydrolysis act as bacterial growth factors. Elucidating the structures of protonated ferric enterobactin may reveal the pathway by which synthetic analogues fulfill bacterial iron requirements. In order to more completely model this potential delivery pathway for ferric iron, as well as to understand the pH dependent structural dynamics of ferric enterobactin, two ligands, (2-hydroxybenzoyl-2-aminoethyl)-bis(2,3-dihydroxybenzoyl-2-aminoethyl)amine (TRENCAMSAM) and (2-hydroxy-3-methoxybenzoyl-2-aminoethyl)-bis(2,3-dihydroxybenzoyl-2- aminoethyl)amine (TRENCAM(3M)SAM), have been synthesized as models for monoprotonated enterobactin. The coordination chemistry of these ligands with Fe3+ and Al3+ has been investigated. Fe[TRENCAMSAM]2- crystallizes in the triclinic space group P1: Z = 1, a = 11.3307(6) A, b = 12.5479(7) A, c = 15.5153(8) A, alpha = 94.513(1) degree, beta = 105.867(1) degree, gamma = 94.332(1) degree. The structure is a two-metal two-ligand dimer supported by mu-oxo bridges from two catecholate moieties. Al[TRENCAMSAM]2- crystallizes in the triclinic space group P1: Z = 2, a = 9.1404(2) A, b = 13.3570(1) A, c = 15.5950(1) A, alpha = 95.711(1) degree, beta = 104.760(1) degree, gamma = 92.603(1) degree. The complex is a monomer with a five-coordinate, square-pyramidal aluminum cation. Al[TRENCAM(3M)SAM]2- crystallizes in the monoclinic space group C2/m: Z = 8, a = 34.244(2) A, b = 11.6206(6) A, c = 21.9890(12) A, beta = 101.478(1) degree. The complex is also a monomer, but with a highly distorted five-coordinate, square-pyramidal aluminum cation coordination sphere. At high pH these complexes do not display a salicylate mode of binding; however, at low pH Al[TRENCAMSAM]2- converts to protonated Al[H3TRENCAMSAM]+, which is a six-coordinate, tris-salicylate complex. Al[H3TRENCAMSAM]+ crystallizes in the triclinic space group P1: Z = 2, a = 11.5475(4) A, b = 12.1681(4) A, c = 12.5094(4) A, alpha = 109.142(1) degree, beta = 104.327(1) degree, gamma = 103.636(1) degree. This is the first catecholamide enterobactin analogue that has been structurally characterized in both a catecholate and salicylate mode of coordination.     

Bromoenterobactins as potent inhibitors of a pathogen-associated, siderophore-modifying C-glycosyltransferase

IroB is a C-glycosyltransferase encoded in the iroA cluster. C-Glucosylation of the bacterial siderophore enterobactin by IroB is a strategy some pathogenic bacteria use to evade the host's innate immunity mediated by lipocalin 2 (Lcn2). Without this modification, enterobactin can be tightly bound by host Lcn2, rendering it ineffective as a siderophore. Therefore, IroB inhibitors could be potential antibiotics against iroA-harboring pathogenic bacteria. We used enterobactin analogues to probe the properties of the active site of IroB and found that enterobactin analogues brominated at the C5 positions of the 2,3-dihydroxybenzoyl rings are potent inhibitors of IroB. This finding could lead to the discovery of effective antibiotics targeting iroA-containing bacteria.     

Corynebactin and a serine trilactone based analogue: chirality and molecular modeling of ferric complexes

Because the hydrolysis of ferric ion makes it very insoluble in aerobic, near neutral pH environments, most species of bacteria produce siderophores to acquire iron, an essential nutrient. The chirality of the ferric siderophore complex plays an important role in cell recognition, uptake, and utilization. Corynebactin, isolated from Gram-positive bacteria, is structurally similar to enterobactin, a well-known siderophore first isolated from Gram-negative bacteria, but contains L-threonine instead of L-serine in the trilactone backbone. Corynebactin also contains a glycine spacer unit in each of the chelating arms. A hybrid analogue (serine-corynebactin) has been prepared which has the trilactone ring of enterobactin and the glycine spacer of corynebactin. The chirality and relative conformational stability of the three ferric complexes of enterobactin, corynebactin, and the hybrid have been investigated by molecular modeling (including MM3 and pBP86/DN density functional theory calculations) and circular dichroism spectra. While enterobactin forms a Delta-ferric complex, corynebactin is Lambda. The hybrid serine-corynebactin forms a nearly racemic mixture, with the Lambda-conformer in slight excess. Each ferric complex has four possible isomers depending on the metal chirality and the conformation of the trilactone ring. For corynebactin, the energy difference between the two possible Lambda conformations is 2.3 kcal/mol. In contrast, only 1.5 kcal/mol separates the inverted Lambda- and normal Delta-configuration for serine-corynebactin. The small energy difference of the two lowest energy configurations is the likely cause for the racemic mixture found in the CD spectra. Both the addition of a glycine spacer and methylation of the trilactone ring (serine to threonine) favor the Lambda-conformation. These structural changes suffice to change the chirality from all Delta (enterobactin) to all Lambda (corynebactin). The single change (glycine spacer) of the hybrid ferric serine-corynebactin gives a mixture of Delta and Lambda, with the Lambda in slight excess.     

Enterobactin- and salmochelin-β-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenic Escherichia coli CFT073

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesis and evaluation of four new siderophore-β-lactam conjugates where the broad-spectrum β-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene glycol (PEG₃) linker. These siderophore-β-lactam conjugates showed enhanced minimum inhibitory concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with uropathogenic E. coli CFT073 demonstrated that the DGE-β-lactams target the pathogen-associated catecholate siderophore receptor IroN. A comparative analysis of siderophore-β-lactams harboring ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced stability to serine β-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the siderophore-β-lactam conjugates revealed cellular morphologies consistent with the inhibition of penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work illuminates the uptake and cell-killing activity of Ent- and DGE-β-lactam conjugates against E. coli and supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of antibiotics in clinical use and targeting pathogenicity.

Siderophore-β-lactam conjugates based on enterobactin and diglucosylated enterobactin enter the periplasm of uropathogenic E. coli CFT073 via the FepA and IroN transporters, and target penicillin-binding proteins.     

Pyochelin, a siderophore of Pseudomonas aeruginosa: physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes

Pseudomonas aeruginosa is an opportunistic pathogen, synthesizing two major siderophores, pyoverdine (Pvd) and pyochelin (Pch), to cover its needs in iron(III). If the high affinity and specificity of Pvd toward iron(III) (pFe = 27.0) was well described in the literature, the physicochemical and coordination properties of Pch toward biologically relevant metals (Fe(III), Cu(II) or Zn(II)) have been only scarcely investigated. We report a thorough physico-chemical investigation of Pch (potentiometry, spectrophotometries, ESI/MS) that highlighted its moderate but significantly higher affinity for Fe(3+) (pFe = 16.0 at p[H] 7.4) than reported previously. We also demonstrated that Pch strongly chelates divalent metals such as Zn(II) (pZn = 11.8 at p[H] 7.4) and Cu(II) (pCu = 14.9 at p[H] 7.4) and forms predominantly 1 : 2 (M(2+)/Pch) complexes. Kinetic studies revealed that the formation of the ferric Pch complexes proceeds through a Eigen-Wilkins dissociative ligand interchange mechanism involving two protonated species of Pch and the Fe(OH)(2+) species of Fe(III). Our physico-chemical parameters supports the previous biochemical studies which proposed that siderophores are not only devoted to iron(III) shuttling but most likely display other specific biological role in the subtle metals homeostasis in microorganisms. This work also represents a step toward deciphering the role of siderophores throughout evolution.     

Targeting virulence: salmochelin modification tunes the antibacterial activity spectrum of β-lactams for pathogen-selective killing of Escherichia coli

New antibiotics are required to treat bacterial infections and counteract the emergence of antibiotic resistance. Pathogen-specific antibiotics have several advantages over broad-spectrum drugs, which include minimal perturbation to the commensal microbiota. We present a strategy for targeting antibiotics to bacterial pathogens that utilises the salmochelin-mediated iron uptake machinery of Gram-negative Escherichia coli. Salmochelins are C-glucosylated derivatives of the siderophore enterobactin. The biosynthesis and utilisation of salmochelins are important for virulence because these siderophores allow pathogens to acquire iron and evade the enterobactin-scavenging host-defense protein lipocalin-2. Inspired by the salmochelins, we report the design and chemoenzymatic preparation of glucosylated enterobactin–β-lactam conjugates that harbour the antibiotics ampicillin (Amp) and amoxicillin (Amx), hereafter GlcEnt–Amp/Amx. The GlcEnt scaffolds are based on mono- and diglucosylated Ent where one catechol moiety is functionalized at the C5 position for antibiotic attachment. We demonstrate that GlcEnt–Amp/Amx provide up to 1000-fold enhanced antimicrobial activity against uropathogenic E. coli relative to the parent β-lactams. Moreover, GlcEnt–Amp/Amx based on a diglucosylated Ent (DGE) platform selectively kill uropathogenic E. coli that express the salmochelin receptor IroN in the presence of non-pathogenic E. coli and other bacterial strains that include the commensal microbe Lactobacillus rhamnosus GG. Moreover, GlcEnt–Amp/Amx evade the host-defense protein lipocalin-2, and exhibit low toxicity to mammalian cells. Our work establishes that siderophore–antibiotic conjugates provide a strategy for targeting virulence, narrowing the activity spectrum of antibiotics in clinical use, and achieving selective delivery of antibacterial cargos to pathogenic bacteria on the basis of siderophore receptor expression.     

硫酸溶液中电化学改性石墨电极对Fe3+/Fe2+的电催化性能与准电容特性

通过循环伏安(CV)、恒电流充放电和电化学阻抗谱(EIS)等测试方法,研究了电化学改性石墨电极对硫酸溶液中Fe3+/Fe2+的电催化性能与准电容特性.结果表明:由于电化学改性石墨电极表面存在大量的含氧活性官能团,使其对Fe3+/Fe2+的氧化还原反应具有极高的电催化性能,并具有可逆反应过程特征.在含有0.5mo·lL-1Fe3++0.5mo·lL-1Fe2+的2.0mo·lL-1H2SO4溶液中,其表观面积比电容是不含铁离子硫酸溶液的1.808倍,达到2.157F·cm-2;同时,铁离子浓度的增大也能够进一步提高其电容量.Fe3+/Fe2+电对的加入增加了充放电时间,有效提高了电化学电容器(EC)的电容存储容量和高功率特性.电化学阻抗谱测试同样证实体系具有明显的电容特性.因此,可以利用电化学改性石墨电极表面的含氧活性官能团和溶液中Fe3+/Fe2+的氧化还原特性来共同储存和释放能量.     

Infrared multiphoton dissociation of the siderophore enterobactin and its Fe(III) complex. Influence of Fe(III) binding on dissociation kinetics and relative energetics

The dissociation pathways of the siderophore enterobactin and its complex with Fe(III) were examined using infrared multiphoton dissociation (IRMPD). Under experimental conditions (pH = 3.5), both compounds' electrospray spectra exhibited exclusively singly-charged anions. The compositions of the dissociation products were characterized by accurate mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The primary dissociation channel for both species was determined to be the loss of one serine group from the precursor molecules. To further investigate the influence of Fe(III) binding on the intramolecular interactions, dissociation kinetics and relative energetics for the loss of this serine group were determined using the focused radiation for gaseous multiphoton energy-transfer (FRAGMENT) method. From the kinetic data, it was found that enterobactin was approximately seven times more reactive than its Fe(III) complex over the range of laser intensities investigated. The relative activation energies, however, exhibited similar values, approximately 7 kcal.mol(-1). These results suggest that at pH = 3.5, Fe(III) interacts with only two of the three serine groups. The results from the present work are believed to be valuable for the characterization of novel siderophores as well as their associated metabolites and synthetic analogues.     

Bacterial iron transport: coordination properties of azotobactin, the highly fluorescent siderophore of Azotobacter vinelandii

Azotobacter vinelandii, a nitrogen-fixing soil bacterium, secretes in iron deficiency azotobactin delta, a highly fluorescent pyoverdin-like chromopeptidic hexadentate siderophore. The chromophore, derived from 2,3-diamino-6,7 dihydroxyquinoline, is bound to a peptide chain of 10 amino acids: (L)-Asp-(D)-Ser-(L)-Hse-Gly-(D)-beta-threo-HOAsp-(L)-Ser-(D)-Cit-(L)-Hse-(L)-Hse lactone-(D)-N(delta)-Acetyl, N(delta)-HOOrn. Azotobactin delta has three different iron(III) binding sites which are one hydroxamate group at the C-terminal end of the peptidic chain (N(delta)-Acetyl, N(delta)-HOOrn), one alpha-hydroxycarboxylic function in the middle of the chain (beta-threo-hydroxyaspartic acid), and one catechol group on the chromophore. The coordination properties of its iron(III) and iron(II) complexes were measured by spectrophotometry, potentiometry, and voltammetry after the determination of the acid-base functions of the uncomplexed free siderophore. Strongly negatively charged ferric species were observed at neutral p[H]'s corresponding to a predominant absolute configuration Lambda of the ferric complex in solution as deduced from CD measurements. The presence of an alpha-hydroxycarboxylic chelating group does not decrease the stability of the iron(III) complex when compared to the main trishydroxamate siderophores or to pyoverdins. The value of the redox potential of ferric azotobactin is highly consistent with a reductive step by physiological reductants for the iron release. Formation and dissociation kinetics of the azotobactin delta ferric complex point out that both ends of this long siderophore chain get coordinated to Fe(III) before the middle. The most striking result provided by fluorescence measurements is the lasting quenching of the fluorophore in the course of the protonation of the ferric azotobactin delta complex. Despite the release of the hydroxyacid and of the catechol, the fluorescence remains indeed quenched, when iron(III) is bound only to the hydroxamic acid, suggesting a folded conformation at this stage, around the metal ion, in contrast to the unfolded species observed for other siderophores such as ferrioxamine or pyoverdin PaA.     

Collision-activated dissociation,infrared multiphoton dissociation,and electron capture dissociation of the <Emphasis Type="Italic">Bacillus anthracis</Emphasis> siderophore petrobactin and its metal ion complexes

Siderophores are high-affinity iron-chelating ligands produced by microorganisms to scavenge vital Fe(3+) from the environment. Thus, siderophores constitute potential therapeutic targets and their structural determination is important for exploiting their therapeutic value. Here, the virulence-associated siderophore petrobactin from Bacillus anthracis was characterized with electron capture dissociation (ECD). Fragmentation of doubly protonated petrobactin was investigated and compared to sustained off-resonance irradiation collision-activated dissociation (SORI CAD) and infrared multiphoton dissociation (IRMPD) of both the singly and doubly protonated species. These experiments demonstrate that ECD provides additional information (complementary bond cleavages) on the structure of petrobactin compared to both SORI CAD and IRMPD. Furthermore, complexes of petrobactin with divalent (Ca(2+), Fe(2+), and Co(2+)) and trivalent (Fe(3+) and Ga(3+)) metal cations were also subjected to SORI CAD and ECD. Again, most structural information was obtained from the ECD spectra. However, significant differences were found in both SORI CAD and ECD of metal complexes, dependent on the nature of the metal ion. Intriguingly, unique behavior, consistent with a recently proposed solution-phase structure, was observed for the highly preferred Fe(3+)-petrobactin complex.     

Electro-nuclear double resonance spectroscopic evidence for a hydroxo-bridge nucleophile involved in catalysis by a dinuclear hydrolase

Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4). The solvent-derived coordination of the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminal water/hydroxide bound to Fe(2+) but no terminal water/hydroxide bound to Fe(3+). The terminal water is lost upon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysis mechanism involving a terminal Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.     

Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16

The bacterium Cupriavidus necator H16 produces a family of linear lipopeptides when grown under low iron conditions. The structural composition of these molecules, exemplified by the main metabolite cupriachelin, is reminiscent of siderophores that are excreted by marine bacteria. Comparable to marine siderophores, the ferric form of cupriachelin exhibits photoreactive properties. Exposure to UV light induces an oxidation of its peptidic backbone and a concomitant reduction of the coordinated Fe(III). Here, we report the genomics-inspired isolation and structural characterization of cupriachelin as well as its encoding gene cluster, which was identified by insertional mutagenesis. Based upon the functional characterization of adenylation domain specificity, a model for cupriachelin biosynthesis is proposed.     

Monoesterase activity of a purple acid phosphatase mimic with a cyclam platform

The synthesis and characterization of a novel dinucleating ligand L (L=4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam) and its μ-oxo-bridged diferric complex [(H(2)L){Fe(III)(2)(O)}(Cl)(4)](2+) are reported. This diiron(III) complex is the first example of a truly functional purple acid phosphatase (PAP) mimic as it accelerates the hydrolysis of the activated phosphomonoester 2,4-dinitrophenyl phosphate (DNPP). The spectroscopic and kinetic data indicate that only substrates that are monodentately bound to one of the two ferric ions can be attacked by a suitable nucleophile. This is, most probably, a terminal iron(III)-bound hydroxide. DFT calculations support this assumption and also highlight the importance of secondary interactions, exerted by the protonated cyclam platform, for the positioning and activation of the iron(III)-bound substrate. Similar effects are postulated in the native enzyme but addressed in PAP mimics for the first time.