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Sulfur mobilization mechanisms for iron-sulfur cluster biosynthesis in Staphylococcus aureus
Sabo, Emily T.
Sabo, Emily T.
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2025
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Abstract
To address the antibiotic resistance crisis, this work investigates essential enzymes in Staphylococcus aureus as targets for antibiotic development. Providing kinetic, structural, and mechanistic understanding of antibiotic targets allows for the strategic design of novel inhibitors. In this work kinetic and spectroscopic methods are utilized to reveal mechanistic and structural insights into the SufS and SufU enzymes, which catalyze sulfur mobilization for the Fe-S cluster biosynthetic SUF pathway, an essential process for S. aureus survival. Chapter 1 reviews the roles of Fe-S clusters in biology, Fe-S clusters in therapeutic development, and the enzymes involved in Fe-S cluster biosynthesis.
In Chapter 2, development of a cysteine desulfurase assay allows for kinetic characterization of SufS from S. aureus (SaSufS). Measurement of Michaelis-Menten kinetic parameters, catalytic impacts from changing pH, and isotope studies confirm the role of important catalytic residues and provide new insights into the mechanism of SaSufS. To build on this work, Chapter 3 utilizes a spectrophotometric method to measure intermediates of the cysteine desulfurase mechanism and quantify the efficacy of novel inhibitors on SaSufS. These inhibitors of SaSufS also inhibited growth for the Methicillin-resistant strain of S. aureus (MRSA). Chapter 4 provides evidence for the second step of the SUF pathway involving the sulfur transfer protein SufU from S. aureus (SaSufU) and its interaction with SaSufS. This was accomplished through substitution of the Zn(II) site in SaSufU with Co(II) allowing for the utilization of techniques including UV-Vis absorbance, EPR, and EXAFS spectroscopies. Together, this work lays the basis for investigation of the SUF pathway as a target for inhibition of S. aureus.
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