Abstract 1412 Binding Thermodynamics in Desferrioxamine D: Cooperative Sites without Cooperativity

Siderophores, critical iron-acquisition molecules for microbes, are synthesized via several pathways, notably the relatively understudied NIS pathways. Critical to the NIS pathways are NIS Synthetase enzymes, including DesD from Streptomyces coelicolor, which are increasingly linked to bacterial virulence, making them appealing drug targets. While structural insights into DesD-cofactor interactions have been established, understanding the binding thermodynamics remains crucial for inhibitor design. In this research, we explored the binding behavior of DesD with ADP and AMP using Isothermal Titration Calorimetry (ITC). Surprisingly, a two-site binding model, with one site exhibiting significantly higher affinity than the other, was observed. Despite DesD being a homodimer, this unexpected binding asymmetry between the two sites challenges the previously established absence of cooperativity in catalytic turnover. Initial assays have established binding conditions, paving the way for future studies exploring substrate recognition by DesD. Structural insights have revealed ATP in both sites but limited substrate presence, underscoring the complexity of DesD's substrate binding. Our current hypothesis proposes a strategic advantage for catalytic efficiency, ensuring an available site for subsequent catalytic events while releasing products. Establishing the structural distinction will be pivotal in directing future studies for effective substrate or inhibitor dosing. This research lays the groundwork for understanding DesD binding thermodynamics, providing insights into substrate recognition. Future endeavors will focus on refining experimental conditions for cleaner data and exploring substrate binding dynamics, essential for effective drug design and catalytic efficiency studies.