The Synthesis and Biochemical Use of N‐Hydroxyl‐N‐(5‐aminopentyl)succinamic acid (HSC) by the Enzyme FslA

Siderophores are small, high affinity iron chelators used by bacteria for resource gathering. Non‐Ribosomal Peptide Synthetases manufacture siderophores using a novel and under‐described chemistry: they create peptide bonds outside of a ribosome, in the process consuming ATP and a high‐energy adenylate intermediate. Beyond basic science, NIS synthetase have a novel fold and chemistry making it a good target for antibiotic design. Despite their interesting function, there are only nine relevant articles on PubMed database about NIS Synthetases. DesD is a model enzyme for this novel family, an enzyme from Streptomyces coelicolor that makes the siderophore desferrioxamine E. The Hoffmann Lab is pursuing a full biochemical characterization of DesD, which will require the appropriate techniques as well as access to the substrates in a pure, soluble form. DesD is an iterative enzyme, meaning it catalyzes multiple bonds on the same substrate. Specifically, DesD catalyzes all of these events: the serial linking of three molecules of N‐Hydroxyl‐N‐(5‐aminopentyl)succinamic acid (HSC) into a linear trimer called desferrioxamine G, and then a macrocylization event linking the ends. The initial binding of two HSC molecules is a very different binding site occupancy than the binding of the linear trimer for the cyclization bond, and any biochemical characterization should quantify this binding and any differences in kinetics. Desferrioxamine G can be commercially bought to study binding of DesD. HSC however, cannot be bought, only synthesized. This project is concerned with the synthesis of HSC and then the use of it in binding and kinetic studies. While the synthesis has been published in the literature, we have made adjustments to improve yield. Additionally, we are optimizing purification via HPLC. This substrate will be used in thermodynamic binding studies via isothermal titration calorimetry and compared to current binding isotherms of the larger substrate. With the study of large substrate and small substrate thermodynamics and kinetics will contribute both to the understanding of NIS synthetases and of iterative enzymes more broadly.T Support or Funding Information This work was supported by a grant from the National Science Foundation (MCB ‐ 1716986) to K.M.H, and by California Lutheran University. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .