Determination of Geochemical Bio-Signatures in Mars-Like Basaltic Environments

Bio-signatures play a central role in determining whether life existed on early Mars. Using a terrestrial basalt as a compositional analog for the martian surface, we applied a combination of experimental microbiology and thermochemical modeling techniques to identify potential geochemical bio-signatures for life on early Mars. Laboratory experiments were used to determine the short-term effects of biota on the dissolution of terrestrial basalt, and the formation of secondary alteration minerals. The chemoorganoheterotrophic bacterium, Burkholderia sp. strain B_33, was grown in a minimal growth medium with and without terrestrial basalt as the sole nutrient source. No growth was detected in the absence of the basalt. In the presence of basalt, during exponential growth, the pH decreased rapidly from pH 7.0 to 3.6 and then gradually increased to a steady-state of equilibrium of between 6.8 and 7.1. Microbial growth coincided with an increase in key elements in the growth medium (Si, K, Ca, Mg, and Fe). Experimental results were compared with theoretical thermochemical modeling to predict growth of secondary alteration minerals, which can be used as bio-signatures, over a geological timescale. We thermochemically modeled the dissolution of the basalt (in the absence of biota) in very dilute brine at 25°C, 1 bar; the pH was buffered by the mineral dissolution and precipitation reactions. Preliminary results suggested that at the water to rock ratio of 1 × 107, zeolite, hematite, chlorite, kaolinite, and apatite formed abiotically. The biotic weathering processes were modeled by varying the pH conditions within the model to adjust for biologic influence. The results suggested that, for a basaltic system, the microbially-mediated dissolution of basalt would result in “simpler” secondary alteration, consisting of Fe-hydroxide and kaolinite, under conditions where the abiotic system would also form chlorite. The results from this study demonstrate that, by using laboratory-based experiments and thermochemical modeling, it is possible to identify secondary alteration minerals that could potentially be used to distinguish between abiotic and biotic weathering processes on early Mars. This work will contribute to the interpretation of data from past, present, and future life detection missions to Mars

Mechanistic and Structural Studies of Salicylate Biosynthesis in Pseudomonas aeruginosa

Iron is an essential element for most pathogenic bacteria. To survive and establish infections in host tissues, these pathogens must compete with the host organism for iron. One strategy is to excrete iron-chelator siderophores with very high affinity to ferric iron in the low iron environment of the host. The phenolate type siderophore, such as pyochelin in Pseudomonas aeruginosa, uses salicylate derived from chorismate as a precursor. Studies have shown that the salicylate activation by adenylation for incorporation to siderophores is associated with the growth and virulence of some pathogens. The inhibition of salicylate biosynthesis, and hence, siderophore production is considered an attractive target for the development of novel antimicrobial agents. In P. aeruginosa, salicylate is derived from chorismate via isochorismate by two enzymes: isochorismate synthase (PchA) and isochorismate-pyruvate lyase (IPL, PchB). PchB eliminates the enolpyruvyl side chain from isochorismate through a biologically unusual pericyclic reaction mechanism. PchB can also perform an adventitious pericyclic reaction that rearranges chorismate to prephenate possibly due to the homology to the E. coli chorismate mutase (CM). The primary contribution to lower the activation energy for enzymatic pericyclic reactions is controversial and may be arise from electrostatic stabilization of the transition state, or conformational stabilization of the reactive substrate. Structural and mutational studies on a key residue, lysine 42, of the active site loop suggest that rate enhancement of the two pericyclic reactions (IPL and CM) performed by PchB results from both the transition state stabilization and the reactive substrate conformation, but the relative contributions are different for each reaction. A mutation with less active site loop mobility, A43P indicates that the loop dynamics is related to catalysis. The I87T structure reveals a larger disordered region compared to the wild type structure, suggesting that conformational mobility may play a role in catalysis. PchA is an isochorismate synthase (ICS) in P. aeruginosa that removes the C4 hydroxyl group and adds a hydroxyl group to C2-chorismate. PchA is homologous to salicylate synthases from Yersinia spp. and Mycobacterium tuberculosis that convert chorismate to salicylate without requirement of an additional lyase such as PchB in P. aeruginosa. A sequence comparison between PchA with salicylate synthases of known structure suggests that two conserved residues are directly involved in the general acid and base chemistry in PchA: K221 as the general base and E269 as the general acid. Replacement of K221 and E269 with alanine respectively led to catalytically inactive enzymes, suggesting that K221 and E269 are critical for ICS catalysis. Preliminary pH dependence data for PchA supports the general acid and base mechanism of PchA catalysis. Two nonconserved residues A375 and D310 were also examined. Replacement of A375 by threonine, the corresponding residue in salicylate synthase, resulted in only residual ICS activity, indicating that A375 is not associated with the IPL-deficiency in PchA. The D310E mutant leads to two additional activities, IPL and CM. The additional activities in three reactions may due to the preferential orientation of substrates in the active site

Structural, Functional and Computational Characterization of Pseudomonas aeruginosa Siderophore Biosynthetic Pathway Accessory Proteins PchB and PvdA

To survive and establish infections in host tissues, pathogens must compete with the host organism for iron. One strategy for iron acquisition is to excrete iron-chelators, called siderophores, with very high affinity to ferric iron. Studies have shown siderophores to be associated with growth and virulence of some pathogens. Inhibition of siderophore production is therefore considered an attractive target for the development of novel antimicrobial agents. This dissertation describes biochemical investigations of two enzymes involved in siderophore production in Pseudomonas aeruginosa, an opportunistic pathogen. PchB catalyzes two pericyclic reactions in a single active site: 1.) an isochorismate-pyruvate lyase reaction (breakdown of isochorismate to salicylate and pyruvate) and 2.) a chorismate mutase reaction (rearrangement of chorismate to prephanate). There is an ongoing debate in the field over the relative contributions of Near Attack Conformations (NAC) and Transition State Stabilization (TSS) to the molecular mechanism of pericyclic reactions. Steady-state kinetics of a K42H-PchB mutant with a pH-dependent charge on position 42 on the active site loop, previously shown to be important for catalysis reveals that lyase and mutase activities require the positive charge at that position for efficient catalysis. Covalent and non-covalent chemical-rescue experiments on mutants deficient of the positive charge at position 42 suggest that the positive charge at the 42 position must be organized within the active site for efficient catalysis. Finally, quantum mechanical/molecular mechanical experiments on wild type and K42H PchB models look at the mechanism of catalysis of the lyase activity in more detail. PvdA is an accessory enzyme to the siderophore pyoverdin biosynthetic pathway of Pseudomonas aeruginosa. PvdA is an N-hydroxylating ornithine hydroxylase: it catalyzes the FAD dependent addition of oxygen to the N5-amine of the ornithine using NADPH as electron donor and molecular oxygen. Here we present two structures of PvdA with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) flavin forms. These are the first two structures of an N-hydroxylating Class B flavoprotein monooxygenase and the first structures of a Class B flavoprotein monooxygenase to contain redox center, electron donor and product/substrate in the active site bound simultaneously

Pericyclic reactions catalyzed by chorismate-utilizing enzymes

This publication was made possible by funds from National Institutes of Health (NIH) Grant P20 RR016475 from the INBRE Program of the National Center for Research Resources and NIH Grants R01 AI77725 and K02 AI093675 from the National Institute for Allergy and Infectious Diseases.One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure-function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST-family (menaquinone, siderophore or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries

Multidentate Ligand Design for the F-Elements

The wide use of the f-elements, including nuclear weapons, nuclear energy and radiopharmaceuticals, has led to the growing unwanted accumulation of the lanthanides and actinides in the environment. The removal of these metals requires the design of highly selective ligands that take advantage of their complex chemistry (wide degree of covalency and high coordination numbers). Additionally, the environments these metals are typically being removed from involve a complicated mixture of other metals and strong counterions. Ligand design for the f-elements requires high selectivity and the formation of stable complexes in a wide range of environments. Ethylene diamine tetraacetic acid (EDTA) is one of the most well-known and widely utilized ligands in coordination chemistry, and hydroxamate-containing ligands are some of the most common in biological chemistry, suggesting a natural combination of EDTA-like backbones with hydroxamate arms. Specifically, the increase from the hexadentate EDTA to the decadentate hydroxamate analog would address the larger coordination numbers of the f-elements. EDTA, however, is too small even for Fe(III), leaving a coordinated water on the Fe, and is far too small for Ln/An(III) ions. Consequently, I synthesized a hydroxamate EDTA analog where the arms are longer by one carbon (ethylenediamine tetrapropionyl hydroxamic acid, EDTPHA). Potentiometric titrations of EDTPHA with La(III), Eu(III), and Lu(III), and a comparison with a synthetic all-carboxylate analog (EDTP) reveal that EDTPHA is powerful ligand for lanthanides competitive with EDTA. During the course of the work I developed a new protecting group for use in hydroxamate synthesis, and the straightforward synthesis of the EDTPHA ligand (utilizing highly pure aza-Michael chemistry) enables future work with additional ligands including EDTA-like ligands as well as siderophore-like peptide ligands to optimize their selectivity for f-element chemistry

The effect of siderophores on the aqueous chemistry of uranium VI: a combined experimental and computational approach

Understanding aqueous uranium VI (UVI) chemistry in alkaline environments (pH >10) is crucial as radioactive waste can be stored and disposed in these conditions. Naturally occurring organic molecules can interact with UVI, modifying its aqueous chemistry and subsequent groundwater facilitated transport. The aim of this research project is to characterise the effect of the (tris)hydroxamate siderophore desferrioxamine B (DFOB) on UVI aqueous chemistry in alkaline solutions. Initially, the physico-chemical properties of UVI precipitates were characterised in solutions containing 42 µM UVI and 0.1 M NaCl. UVI formed 640 ± 111 and 837 ± 142 nm diameter Na6U7O24 precipitates at pH 10.5 and 11.5 respectively. These were usually physically immobilised in quartz sand columns. When ≥130 µM DFOB was simultaneously added with UVI to pH 11.5, 0.1 M NaCl solutions, UVI quantitatively passes through 0.2 µm filter membranes. This could be due to the formation of an aqueous UVI-DFOB complex as observed below pH 10. To further explore complex formation, a density functional theory protocol was established. The protocol predicts the stability constants (log β) of UVI-organic ligand complexes with root mean square deviation of 1.19 log β units after calibration against experimental data collected in acidic solutions. The relative stability series for UVI complexes with key siderophore functional groups calculated using the fitting equation is: α-hydroxycarboxylate bound via the α-hydroxy and carboxylate groups (log β110 = 17.08), α-hydroxyimidazolate (log β110 = 16.55), catecholate (log β110 = 16.43), hydroxamate (log β110 = 9.00), hydroxy-phenyloxazolonate (log β110 = 8.43) and α-aminocarboxylate (log β110 = 4.73). Finally the DFT protocol was adapted so that the stability of UVI-hydroxamate complexes can be approximated at pH 11.5. This suggests DFOB complexes in a monodentate fashion via one hydroxamate group. These results highlight the significant effect siderophores can play on aqueous UVI chemistry.Open Acces

Using iron to catch a ride - synthetic siderophores as molecular 'Trojan Horses' to visualize and treat MDR bacterial pathogens

The rise in multidrug-resistant, bacterial infections, together with a shallow industrial discovery pipeline, urgently calls for novel diagnostic and therapeutic strategies. Bacterial cells, particularly Gram-negative pathogens with their double-layered cell wall, closely resemble a fortress that restricts the accumulation of small molecules. However, microbial transporters ensure a sufficient nutrient supply during infection of a host organism and act as gateways into the pathogen, e.g. for ferric iron, which plays a crucial role in microbial metabolism and growth. Siderophores, small bacterial molecule chelators, sequester Fe3+ from host proteins and are transported by bacterial, TonB-dependent transporters (TBDTs). Like a molecular “Trojan Horse”, synthetic siderophore mimics can hijack the siderophore transport system and actively translocate diagnostic or therapeutic payloads over the impervious bacterial membrane and accumulate at their site of action. This thesis expanded and evaluated the potential of synthetic and natural siderophores for the visualization and antibiotic therapy of MDR bacteria in cellular and in vivo. The structure of the DOTAM triscatecholate siderophore was adapted for an application as a bacteria-specific, gallium-68 labeled PET tracer for the detection of bacterial infections in vivo. Two tracers showed good in vitro, radiochemical and pharmacokinetic properties in vivo and selectively accumulated at the site of infection vs. a site of sterile inflammation. Similarly, chemiluminescent dioxetanes were attached to siderophores to yield a panel of siderophore dioxetane probes that detected Gram-positive and Gram-negative bacterial pathogens. The best compound exhibited superior stability in bacterial supernatant, detected low bacterial counts and even intracellular bacteria in infected lung epithelial cells. In an attempt to enhance the accumulation in Gram-negative bacteria and thus restore the activity of antibiotics used only against Gram-positive bacteria (e.g. lipopeptides, ansamycins, macrolides), chelators were conjugated via covalent and cleavable linker systems, to yield potent drug conjugates. Studies on siderophore receptor mutants of E. coli and P. aeruginosa, including transcriptomic and proteomic investigations, contributed information on the involved siderophore transporters as well as on the mechanistic response upon siderophore and conjugate addition. Peptide siderophore conjugates that target the TonB-dependent transport of ferric chelates in Pseudomonas successfully inhibited bacterial growth. This proof-of-concept established TonB as a novel target in antimicrobial therapy. The design, synthesis and biological evaluation of novel diagnostic and therapeutic siderophore conjugates represents an important milestone towards a clinical usage of this approach against MDR ESKAPE bacteria

Structural basis of antibacterial peptide transport across membranes

Microcins are gene encoded antibacterial peptides secreted by enterobacteria in the gastrointestinal tract and play an important role in the control of bacterial populations. They present an attractive prospect in our effort to minimize the problem of bacterial drug resistance. Microcin J25 (MccJ25) is a 2 kDa plasmid encoded, ribosomally synthesized antimicrobial peptide comprised of 21 amino acid residues. MccJ25 undergoes post-translational modification and has a unique lasso structure. McjD, an ABC exporter, confers immunity to the producing strains by exporting the mature MccJ25 out of the cell. Studies have been designed to look into the transport mechanism of this peptide, which uses the siderophore receptor FhuA and ABC transporter McjD. MccJ25 uses the Trojan horse strategy by hijacking the iron import machineries as a mode of transport into the cell and acts as a transcription inhibitor by binding to RNA polymerase. Iron is an important nutrient for bacteria cell survival. To date, there is limited structural evidence on the import and extrusion mechanism of this antimicrobial peptide in Gram-negative bacteria. We have obtained a high-resolution structure of MccJ25 with its outer membrane receptor FhuA at 2.3 Å. FhuA is monomeric 22-strand antiparallel ß-barrel protein with the N-terminal domain folded inside to form a plug domain. MccJ25 binds to FhuA with hydrogen bonds and hydrophobic interactions with the extracellular loops of FhuA and its plug domain. We have also identified key residues that might play a role in MccJ25 translocation. Overall the structure provides information on how MccJ25 hijacks the iron uptake pathway to get into bacteria. Ligand binding studies and biochemical analysis demonstrate the functionality of McjD and its interaction with its natural ligand, MccJ25. The high substrate specificity and known cavity make McjD an excellent model for interaction studies.Open Acces

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference