Siderophores are small, iron chelating molecules produced by many bacteria to help meet the iron requirements of the cell. Multiple metabolic functions require iron as it serves as a cofactor in many enzymes and cellular processes. However, in the presence of oxygen and at physiologic pH, iron forms insoluble ferric complexes that cause the nutrient to be unavailable to bacterial cells. Siderophores alleviate this limitation by chelating the ferric iron, rendering it soluble and available for uptake. One group of microorganisms known for their ability to produce siderophores is the rhizobia. These bacteria are characterized both by their formation of symbiotic relationships with leguminous plants and their ability to fix atmospheric nitrogen. Rhizobium leguminosarum ATCC 14479, which infects the red clover Trifolium pratense, was found to produce a trihydroxamate siderophore. Purification and chemical characterization identified this siderophore as Vicibactin that has been found to be produced by other rhizobial strains.

Iron is necessary for many cellular processes such as the electron transport chain and gene regulation. However, most iron on earth is found in insoluble iron-hydroxide complexes. In addition, iron is tightly sequestered in the human body by proteins such as transferrin, making it unavailable for pathogens. In order to overcome these limitations bacteria have evolved siderophores. Siderophores are low molecular weight compounds that bind ferric iron with a high affinity. Staphylococcus aureus is an important human pathogen that is known to produce at least four siderophores, and these siderophores contribute to its virulence. S. aureus strain H372 was found to produce a siderophore that was a carboxylate type, hydrophilic, and contained ornithine. These properties were similar to the known siderophore staphyloferrin A. However, the probable molecular weight was 658, which is different from known staphylococcal siderophores.

Iron is essential for life and growth in most organisms. Although it is abundant, iron exists mostly as insoluble iron-oxyhydroxide. Bacteria secrete siderophores to chelate iron and transport it into the cell via specific outer membrane receptors. The FhuA receptor protein transports ferrichrome, a siderophore produced by Ustilago sphaerogena. We determined the binding affinity of variants from the conserved 'lock region' of FhuA and also created and characterized variants of the highly conserved R452 to determine its role in ferrichrome transport. We hypothesize that during transport the plug domain of FhuA does not leave the barrel; rather it undergoes a conformational change to form a channel. We mutated selected amino acids to cysteine to form disulfide bonds to tether the plug, preventing its displacement or unfolding during transport. The tetra-cysteine mutant 72/615/109/356C was able to bind and transport radiolabeled ferrichrome. One double-cysteine mutant, 104/149C, was purified for crystallization.

Iron is an essential nutrient for most bacteria because enzymes like nitrate reductase and cytochromes use it as a cofactor. However, in most aerobic, neutral pH environments, iron is essentially insoluble and not easily available for bacteria to use. Many bacteria respond to this problem by releasing small organic compounds called siderophores that bind and effectively solubilize iron so that it can be transported into the cell for growth. The focus of this study was to learn more about the iron acquisition and especially the transport of iron by the soil bacterium Rhodococcus erythropolis. To fulfill this aim, mutant strains of the bacteria were screened for those that overproduce siderophore. Often, a bacterium will over produce siderophore to compensate for a defect in transport. One such mutant, R187-12, was further analyzed by cloning the region of the chromosome containing the defective gene responsible for over production of siderophore into a plasmid vector. The DNA sequence of this region was determined and analyzed for the presence of similar genes encoding transport proteins.

This thesis consists of two chapters, each of which is a unique research project. Chapter 1 is focused on the synthesis and biological evaluation of novel sideromycin antibiotics. Sideromycins are bifunctional “Trojan Horse” molecules that have an iron chelator “siderophore” moiety covalently bound to an antibiotic. Such molecules exploit existing bacterial mechanisms for obtaining iron from their environment. Antibiotics that would typically not pass Gram-negative membranes are allowed access via siderophore transporter proteins. This project utilized a siderophore that has not previously been used in this capacity. The synthesis and biological evaluation of multiple sideromycin conjugates is reported. Chapter 2 describes the chemical synthesis of coumarin natural products using a synthetic process recently developed in the Magolan laboratory that enables the efficient prenylation of phenols. These natural products are molecules of biological interest in various capacities but are rare and difficult to isolate from their plant sources. They have also previously been cumbersome to make via chemical synthesis. The chemistry described herein constitutes an inexpensive and efficient process to produce these compounds that is superior to previously known methods. / Thesis / Master of Science (MSc) / This thesis is divided into two chapters. The first chapter is focused on the development of new sideromycin antibiotics. Sideromycins are “Trojan Horse”-like antibiotics that exploit the mechanisms of Gram-negative bacteria for obtaining iron, an essential nutrient, to enable antibiotic entry. This chapter details the synthesis of molecules that attach functionalities called “siderophores” to antibiotics, enabling them to be “smuggled” into Gram-negative microbes. This project uses a siderophore not previously utilized in sideromycin research. The second chapter is focused on the chemical synthesis of rare natural products that are phenols with prenyl substituents. Many such compounds are plant-derived and have potential for biomedical use. However, difficulty in isolating them makes them prohibitively expensive in the purity and quantity required for research. They are also challenging to make synthetically. This chapter details the application of a recently discovered process in the Magolan laboratory to synthesize coumarin-containing prenylated phenolic natural products.

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SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector. / Master of Science in Life Sciences