Large and Small Substrate Binding to DesD via ITC Assay Development

Abstract

Desferrioxamines (“dfo”s) are iron‐chelating compounds which have been used medicinally in cases of iron overload. The synthesis of dfoE involves an enzyme called DesD which is a dimer that has an active site on each chain, but the mechanism of it binding both large or small substrates is unknown. We are studying DesD as a model enzyme for the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway and as a potential new drug target. The purpose of this research is to analyze the co‐factor specificity and large substrate verse small substrate specificity of DesD by comparing substrate dissociation constants to analog dissociation constants (KD, where a smaller number correlates to tighter binding). Future drugs will have to out‐compete the substrates in binding, so we will use these studies to determine which substrate components contribute to maximum binding. Techniques in analytical chemistry will be used to regenerate deferoxamines that were bound to cofactors in previous studies with the DesD enzyme. The binding properties of DesD were explored in a 25% glycerol Isothermal Titration Calorimetry (ITC) buffer. Prior experimentation with this protein was conducted in a buffer that resulted in precipitation of DesD out of solution, thus prompting the need for repeated binding assays in a buffer that maintains synthase solubility. GraphPad Prism 8.0 will be used to analyze the KD and occupancy of DesD. High Performance Liquid Chromatography (HPLC) will be used to separate deferoxamines from substrates they were bound to. An Ocean Optics Spectrometer will be used to quantify the deferoxamines by pairing these substrates to a metal compound that will allow us to identify its absorbance and determine if isolation was effective.We confirmed previous results that the KD of DesD binding to ATP is in the M range, compared to non‐ATP cofactors which result in a KD in the M range. Future studies will compare dfoG (substrate) binding to DesD compared to analog dfoB or product dfoE, and also explore smaller substrates, hydroxysuccinylcadaverine (HSC), vs. analogs. Troubleshooting runs have indicated that isolation of the deferoxamines is feasible. It is expected that substrates like dfoG will be separated from ATP and ADP cofactors. We have confirmed that DesD has a strong binding preference for ATP over non‐ATP cofactors in a buffer that maximizes solubility. This data indicates that the phosphates are essential to tight binding in any future competitive inhibitors. Regeneration of each deferoxamine appeared to be a cost‐effective assay that will allow for these compounds to be used in later experimentation. Future work will expand our understanding of critical regions in the binding site, and aid in developing new inhibitors that could block the NIS pathways and be lead drugs for a new class of antibiotics.