<br/>The ability of Desferrioxamine D (DesD) to catalyze the sequential formation of non-ribosomal peptide bonds is unique and understudied. Interestingly, DesD catalyzes multiple amide bond formations on the monomeric substrate N-hydroxy, N-succinyl cadaverine (HSC) to give a dimer, a trimer Desferrioxamine G (dfoG), and ultimately the macrocyclic siderophore known as Desferrioxamine E (dfoE). Uncovering the biophysical details of this synthesis remains of interest given that the target molecule is a clinically useful iron-chelating agent and that inhibition of DesD is a possible strategy for combating pathogenic bacteria.<br/>Loss of function variants were used in kinetic and thermodynamic assays to evaluate the role of individual amino acids in siderophore synthesis. Variants were overexpressed in E. coli, purified over a Ni-affinity column, and buffer exchanged over a desalting column. Kinetic and thermodynamic assays were performed on a Malvern PEAQ isothermal titration calorimeter. X-ray crystallographic structures were also pursued for each variant to visualize conformational changes in the active site. Analysis of the kinetics and thermodynamics of dfoG’s conversion to dfoE has implicated three residues critical to native enzyme function: Arg306, Glu467, and His443. Upon removal of Arg306, complete loss of activity is observed while Glu467 and His443 cause a significant reduction in kcat, possibly reflecting a two-step mechanism.<br/>In the absence of commercially available HSC, a Gabriel amine synthesis that was reported in the literature by others has been reproduced on a multi-gram scale. Regarding noteworthy modifications, we find that: 1) elevated H2 pressure and Pd(OH)2 give a more reliable removal of the benzyl and carbamate protecting groups in the precursor to HSC, and 2) for final purification standard silica gel chromatography in absolute methanol is preferable to semi-preparative HPLC and gradient elution on a C-18 column. HSC prepared under these conditions gives colorless material that is analytically pure and of sufficient quantity for use in kinetic and thermodynamic assays.<br/>Access to synthetic HSC will facilitate the ITC analyses of all DesD variants with monomer as substrate and test our hypothesis of cooperativity and half occupancy as observed in binding studies. We will also explore the possibility of a two-step mechanism by analyzing reaction mixtures via HPLC after catalytic turnover to confirm the intermediacy of both dimer and trimer.

Studies on the mechanism, kinetics, and thermodynamics of desferrioxamine D

ACS Spring 2022

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