| dc.description.abstract | Metal acquisition is a necessity for all cells as metals are essential for the function of numerous metalloproteins and metalloenzymes. Bacterial pathogens employ several strategies for obtaining necessary metal including the direct import of metal ions, the import of metal-containing heme groups, and the secretion of biosynthesized small molecule metallophores to capture metals from the surrounding environment. Metallophore pathways are virulence factors that allow effective competition within the human host and the establishment of infection. They represent viable targets for the development of new antibiotic therapies and an understanding of the production and use of each metallophore contributes to a broader understanding of bacterial pathogenesis. In 2015, Gi and Choi proposed the existence of a novel nicotianamine-like metallophore produced by two enzymes encoded in a four gene operon in Pseudomonas aeruginosa. Nicotianamines are plant-derived metallophores and P. aeruginosa biosynthesizes two well-studied metallophores, pyoverdin and pyochelin, making this hypothesis surprising. In 2016, Ghssein et. al. described a homologous system in Staphylococcus aureus producing a metallophore they named staphylopine biosynthesized by two primary enzymes, a nicotianamine synthase and an opine dehydrogenase. Following heterologous expression of these two orthologous enzymes from the P. aeruginosa operon, a plate-reader screening assay was used to determine the substrates. With substrates known, the predicted structure of pseudopaline was confirmed by mass spectrometry. Pseudopaline is uses L-histidine and -ketoglutarate as substrates in constrast to staphylopine which incorporates D-histidine and pyruvate. Steady-state kinetic parameters for the nicotianamine synthase in S. aureus and P. aeruginosa demonstrated slow kcat values of 1.79 0.02 and 1.07 0.02 min-1 and Km values of 13.0 0.7 and 5.4 0.4 µM respectively. Steady-state parameters had not been previously established for any nicotianamine synthase. This work is detailed in chapter two. The recognition of the enzymatic determinants of opine metallophore production allowed targeted bioinformatics analyses that revealed the presence of like operons in disparate bacterial species living in diverse environments. These analyses are described in chapter one. Opine dehydrogenase enzymes had been structurally characterized from two species (the soil bacterium Arthrobacter sp. 1C and the marine scallop Pecten maximus) prior to this work. Yersinia pestis has a homologous opine metallophore-producing operon. Heterologous expression of the Y. pestis opine dehydrogenase revealed a specificity for L-histidine and pyruvate, although expression of the Y. pestis nicotianamine synthase was unsuccessful and the structure of yersinopine remains presumptive. Y. pestis opine dehydrogenase enriched with selenomethionine crystallized and single wavelength anomalous dispersion (SAD) X-ray diffraction data were collected allowing structure solution. The Y. pestis opine dehydrogenase structure was then used as a molecular replacement model in the structure solution for data sets collected from S. aureus and P. aeruginosa opine dehydrogenase crystals, expanding the number of determined opine dehydrogenase structures in the Protein Data Bank from two to five. A steady-state comparison of these three opine dehydrogenases revealed differences in substrate specificity. S. aureus and Y. pestis use pyruvate and NADPH while P. aeruginosa uses -ketoglutarate and can use either NADH or NADPH. These analyses are described in chapter three. Opine dehydrogenases perform condensation and reduction steps on the prochiral -carbon of an -ketoacid and specifically generate one stereocenter, (R) or (S), depending on their structural family. Opine metallophore-producing opine dehydrogenases are in the family that produce (R) stereocenters. We demonstrated that the S. aureus and P. aeruginosa opine dehydrogenase reactions are reversible and catalyze the reverse reaction only with the (R) diastereomer of staphylopine and pseudopaline respectively. No catalysis was seen in the presence of the (S) diastereomer. In the previously published opine dehydrogenase structures, NADP+ was bound with at least partial electron density, but a structure with bound substrates had not been solved. Structures of P. aeruginosa opine dehydrogenase were solved with both the (R) and (S) diastereomers of pseudopaline. Interestingly, while complete density of (S)-pseudopaline was visualized at 1.64 Å, (R)-pseudopaline was progressively hydrolyzed by the cystal demonstrating reverse catalysis in the X-ray structures. Transient state kinetic analysis of S. aureus opine dehydrogenase revealed that product release was the rate-limiting step of catalysis. These data are described in chapter four. Opine metallophores represent a new class of bacterial metallophore produced by a nicotianamine synthase and an opine dehydrogenase. Nicotianamine synthases remain poorly characterized and future work on the structure and kinetics of this enzyme family will be necessary to provide a mechanistic understanding of function. Our analysis of opine dehydrogenases from S. aureus and P. aeruginosa provides an excellent structure/function analysis and is the foundation for advanced kinetic analyses. These data, along with the expanding analysis of the functional roles of opine metallophore pathways in vivo, are leading toward a global model describing the function of these systems. | |
