The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal [known as Neutrophil Gelatinase Associated Lipocalin, Siderocalin, Lipocalin 2] sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography, and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH sensitive mechanism. Since catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal:catechol:iron complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal:siderophore interactions, but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Systemic anthrax, caused by inhalation or ingestion of Bacillus anthracis spores, is characterized by rapid microbial growth stages that require iron. Tightly bound and highly regulated in a mammalian host, iron is scarce during an infection. To scavenge iron from its environment, B. anthracis synthesizes by independent pathways two small molecules, the siderophores bacillibactin (BB) and petrobactin (PB). Despite the great efficiency of BB at chelating iron, PB may be the only siderophore necessary to ensure full virulence of the pathogen. In the present work, we show that BB is specifically bound by siderocalin, a recently discovered innate immune protein that is part of an antibacterial iron-depletion defense. In contrast, neither PB nor its ferric complex is bound by siderocalin. Although BB incorporates the common 2,3-dihydroxybenzoyl iron-chelating subunit, PB is novel in that it incorporates the very unusual 3,4-dihydroxybenzoyl chelating subunit. This structural variation results in a large change in the shape of both the iron complex and the free siderophore that precludes siderocalin binding, a stealthy evasion of the immune system. Our results indicate that the blockade of bacterial siderophore-mediated iron acquisition by siderocalin is not restricted to enteric pathogenic organisms and may be a general defense mechanism against several different bacterial species. Significantly, to evade this innate immune response, B. anthracis produces PB, which plays a key role in virulence of the organism. This analysis argues for antianthrax strategies targeting siderophore synthesis and uptake.bacillibactin ͉ Bacillus anthracis ͉ petrobactin ͉ siderocalin

Bacillibactin and enterobactin are hexadentate catecholate siderophores produced by bacteria upon iron limitation to scavenge ferric ion and seem to be the ultimate siderophores of their two respective domains: Gram-positive and Gram-negative. Iron acquisition mediated by these trilactone-based ligands necessitates enzymatic hydrolysis of the scaffold for successful intracellular iron delivery. The esterases BesA and Fes hydrolyze bacillibactin and enterobactin, respectively, as well as the corresponding iron complexes. Bacillibactin binds iron through three 2,3-catecholamide moieties linked to a tri-threonine scaffold via glycine spacers, whereas in enterobactin the iron-binding moieties are directly attached to a tri-l-serine backbone; although apparently minor, these structural differences result in markedly different iron coordination properties and iron transport behavior. Comparison of the solution thermodynamic and circular dichroism properties of bacillibactin, enterobactin and the synthetic analogs d-enterobactin, SERGlyCAM and d-SERGlyCAM has determined the role of each different feature in the siderophores' molecular structures in ferric complex stability and metal chirality. While opposite metal chiralities in the different complexes did not affect transport and incorporation in Bacillus subtilis, ferric complexes formed with the various siderophores did not systematically promote growth of the bacteria. The bacillibactin esterase BesA is less specific than the enterobactin esterase Fes; BesA can hydrolyze the trilactones of both siderophores, while only the tri-l-serine trilactone is a substrate of Fes. Both enzymes are stereospecific and cannot cleave tri-d-serine lactones. These data provide a complete picture of the microbial iron transport mediated by these two siderophores, from initial recognition and transport to intracellular iron release.

The hydrogenation of alkenes by heterogeneous catalysts has been studied for 80 years. The foundational mechanism was proposed by Horiuti and Polanyi in 1934 and consists of three steps: (i) alkene adsorption on the surface of the hydrogenated metal catalyst, (ii) hydrogen migration to the β-carbon of the alkene with formation of a σ-bond between the metal and α-C, and finally (iii) reductive elimination of the free alkane. Hundreds of papers have appeared on the topic, along with a number of variations on the Horiuti–Polanyi mechanism. The second step is highly reversible, leading to extensive deuterium–hydrogen exchange when D2(g) is used. This paper describes the investigation of gas-phase reactions between deuterium and 1-butene using a supported palladium catalyst under ambient laboratory conditions and how the results are consistent with the Horiuti–Polanyi mechanism. An Excel spreadsheet for modeling the extent and distribution of deuteration within butane-d x is described. Interested readers could develop a laboratory or research experience based on results presented here. The results are also suitable for inclusion in an upper-division chemistry course in which organometallic chemistry or reaction mechanisms involving heterogeneous catalysts are discussed. The catalyst tubes are inexpensive and easy to construct. Analysis of the butane produced by 1H NMR and GC–MS leads to numerous conclusions in support of the Horiuti–Polanyi mechanism.

Iron is required for virulence of most bacterial pathogens, many of which rely on siderophores, smallmolecule chelators, to scavenge iron in mammalian hosts. As an immune response, the human protein Siderocalin binds both apo and ferric siderophores in order to intercept delivery of iron to the bacterium, impeding virulence. The introduction of steric clashes into the siderophore structure is an important mechanism of evading sequestration. However, in the absence of steric incompatibilities, electrostatic interactions determine siderophore strength of binding by Siderocalin. By using a series of isosteric enterobactin analogs, the contribution of electrostatic interactions, including both chargecharge and cation-π, to the recognition of 2,3-catecholate siderophores has been deconvoluted. The analogs used in the study incorporate a systematic combination of 2,3-catecholamide (CAM) and N-hydroxypyridinonate (1,2-HOPO) binding units on a tris(2-aminoethyl)amine backbone, [tren (CAM) m (1,2-HOPO) n where m = 0, 1, 2, or 3 and n = 3 -m]. The shape complementarity of the synthetic analog series was determined through small molecule crystallography and the binding interactions investigated through a fluorescence-based binding assay. These results were modeled and correlated through ab initio calculations of the electrostatic properties of the binding units. Although all the analogs are accommodated in the binding pocket of Siderocalin, the ferric complexes incorporating decreasing numbers of CAM units are bound with decreasing affinities (K d = >600, 43, 0.8 and 0.3 nM for m = 0-3). These results elucidate the role of electrostatics in the mechanism of siderophore recognition by Siderocalin.

SUMMARY Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding co-purified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.

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The innate immune system, antibacterial protein Siderocalin (Scn) binds ferric carboxymycobactin (CMB) and also several catecholate siderophores. While the recognition of catecholates by Scn has been thoroughly investigated, the binding interactions of Scn with the full spectrum of CMB isoforms have not been studied. Here we show that Scn uses different binding modes for the limited subset of bound CMB isoforms, resulting in a range of binding affinities which are much weaker than other siderophore targets of Scn. Understanding the binding interaction between Scn and CMBs provides clues for the influence of Scn on mycobacterial iron acquisition.