Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of (hvKP). In contrast to classical opportunistic strains of, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. heterologous production and reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.

Iron is a vital mineral nutrient required by virtually all life forms to prosper; pathogenic bacteria are no exception. Despite the abundance of iron within the human host, highly regulated iron physiology can result in exceedingly low levels of iron bioavailable to prospective invading bacteria. To combat this scarcity of iron, many pathogenic bacteria have acquired specific and efficient iron acquisition systems, which allow them to thrive in iron-deficient host environments. One of the more prominent bacterial iron acquisition systems involves the synthesis, secretion, and reuptake of small-molecule iron chelators known as siderophores. Aerobactin, a citrate-hydroxamate siderophore originally isolated nearly 50 years ago, is produced by a number of pathogenic Gram-negative bacteria. Aerobactin has recently been demonstrated to play a pivotal role in mediating the enhanced virulence of a particularly invasive pathotype of Klebsiella pneumoniae (hvKP). Toward further understanding of this key virulence factor, we report the structural and functional characterization of aerobactin synthetase IucA from a strain of hvKP. The X-ray crystal structures of unliganded and ATP-bound forms of IucA were solved, forming the foundation of our structural analysis. Small angle X-ray scattering (SAXS) data suggest that, unlike its closest structurally characterized homologues, IucA adopts a tetrameric assembly in solution. Finally, we employed activity assays to investigate the substrate specificity and determine the apparent steady-state kinetic parameters of IucA.

The critical role that iron plays in many biochemical processes has led to an elaborate battle between bacterial pathogens and their hosts to acquire and withhold this critical nutrient. Exploitation of iron nutritional immunity is being increasingly appreciated as a potential antivirulence therapeutic strategy, especially against problematic multi-drug resistant Gram-negative pathogens such as Acinetobacter baumannii. To facilitate iron uptake and promote growth, A. baumannii produces a nonribosomally synthesized peptide siderophore called acinetobactin. Acinetobactin is unusual in that it is first biosynthesized in an oxazoline form called pre-acinetobactin that spontaneously isomerizes to the final isoxazolidinone acinetobactin. Interestingly, both isomers can bind iron and both support growth of A. baumannii. To address how the two isomers chelate their ferric cargo and how the complexes are used by A. baumannii, structural studies were carried out with the ferric acinetobactin complex and its periplasmic siderophore binding protein BauB. Herein, we present the crystal structure of BauB bound to a bis-tridentate (Fe3+L2) siderophore complex. Additionally, we present binding studies that show multiple variants of acinetobactin bind BauB with no apparent change in affinity. These results are consistent with the structural model that depicts few direct polar interactions between BauB and the acinetobactin backbone. This structural and functional characterization of acinetobactin and its requisite binding protein BauB provides insight that could be exploited to target this critical iron acquisition system and provide a novel approach to treat infections caused by this important multi-drug resistant pathogen.

A set of novel lithium Schiff base cluster compounds has been synthesised and characterised for the first time and tested as electron injectors in OLED devices. Their electrical, electronic, thermal and optical properties have been investigated and compared with the industry standards LiF and lithium quinolinolate (LiQ). Amongst the compounds tested, lithium 2-((o-tolylimino)methyl) phenolate was found to enhance the efficiency of OLEDs by 69% compared to LiF and 15% compared to LiQ. The same electron injector was found to extend the lifetimes of OLEDs by six-fold compared to LiF and 4.3-fold compared to LiQ respectively. The crystal structure of the parent compound, lithium 2-((phenylamino)methyl)phenolate reveals that the compound is tetrameric in contrast to hexameric LiQ. Substituting the methyl group with fluorine causes a remarkable depression of the HOMO and LUMO levels by up to 1.2 eV. Analysis of current density vs. voltage characteristics of single-layer devices for Li-Al/electron injector/Li-Al and Al/electron injector/Al reveals that both sets of devices are operating as electron-only devices indicating that the formation of free lithium is the cause of enhanced electron injection, but either the energetic aluminium atoms (as proposed previously by other workers) or energetic lithium complexes on an aluminium surface (as we have demonstrated in this paper) are all that is required for efficient electron injection.

Two new phases of zirconium tetrakis(8-hydroxyquinolinolate) (Zrq 4) have been synthesised and characterised by single crystal X-ray diffraction. Their electrical, electronic, optical and thermal properties have been studied. Their electron transporting characteristics have been investigated in organic light emitting devices where the two phases show remarkable differences in performance. One of the forms (designated a-Zrq 4) gives significantly lower operating voltage, higher efficiencies and longer lifetime than the other (designated b-Zrq 4) in organic light emitting devices.

Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N 6 -acetyl-N 6hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with sitedirected mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

Acquiring sufficient quantities of iron to support survival is often a critical limitation for pathogenic bacteria. To meet this demand, bacteria have evolved unique strategies to scavenge iron and circumvent the nutritional immunity exerted by their hosts. One common strategy, which is often a key virulence factor for bacterial pathogens, involves the synthesis, secretion, and reuptake of iron chelators known as siderophores. In vitro and in vivo studies have demonstrated that the siderophore aerobactin is critical for virulence in the hypervirulent pathotype of Klebsiella pneumoniae (hvKP). Given the high rate of multidrug resistance in K. pneumoniae, and in light of the ever-increasing demand for novel Gram-negative therapeutic targets, we identified aerobactin production as a promising antivirulence target in hvKP. Herein, we describe the development of a high-throughput biochemical assay for identifying inhibitors of the aerobactin synthetase IucA. The assay was employed to screen ~110,000 compounds across several commercially available small-molecule libraries. IucA inhibitors with activity at micromolar concentrations were identified in our screening campaigns and confirmed using secondary orthogonal assays. However, the most potent compounds also exhibited some properties commonly observed with promiscuous/nonspecific inhibitors, including incubation time and target enzyme concentration dependence, as well as the potential to antagonize unrelated enzymes.

The mononuclear macrocyclic Pd(II) complex cis-[PdCl2([9]aneS3)] ([9]aneS3 = 1,4,7-trithiacyclo-nonane) converts at 44 kbar into an intensely coloured chain polymer exhibiting distorted octahedral coordination at the metal centre and an unprecedented [1233] conformation for the thioether ligand. The evolution of an intramolecular axial sulfur-metal interaction and an intermolecular equatorial sulfur-metal interaction is central to these changes. High-pressure crystallographic experiments have also been undertaken on the related complexes [PtCl2([9]aneS3)], [PdBr2([9]aneS3)], [PtBr2([9]aneS3)], [PdI2([9]aneS3)] and [PtI2([9]aneS3)] in order to establish the effects of changing the halide ligands and the metal centre on the behaviour of these complexes under pressure. While all complexes undergo contraction of the various interaction distances with increasing pressure, only [PdCl2([9]aneS3)] undergoes a phase change. Pressure-induced I...I interactions were observed for [PdI2([9]aneS3)] and [PtI2([9]aneS3)] at 19 kbar, but the corresponding Br...Br interactions in [PdBr2([9]aneS3)] and [PtBr2([9]aneS3)] only become significant at much higher pressure (58 kbar). Accompanying density functional theory (DFT) calculations have yielded interaction energies and bond orders for the sulfur-metal interactions.