Modular Inhibitor Approach to Map the Catalytic Trajectory of an Iterative Siderophore Synthetase, DesD

High Resolution Image Download MS PowerPoint Slide Under iron limiting conditions, bacteria biosynthesize and secrete small molecule iron chelators, siderophores, to scavenge this essential metal. Siderophores are biosynthesized by nonribosomal peptide synthetases (NRPS) or NRPS independent siderophore (NIS) synthetases, the latter of which are significantly less studied. Streptomyces spp . utilizes an iterative NIS synthetase, DesD, to produce desferrioxamine type siderophores through dimerization, trimerization, and (in some cases) macrocyclization of monomers such as N 1 -hydroxy- N 1 -succinyl cadaverine (HSC) and N 1 -hydroxy- N 1 -acetyl cadaverine (HAC). Prior work has utilized an acyl-sulfamoyl adenosine (AMS) inhibitor of monomeric HSC (HSC-AMS) to interrogate the initial adenylation reaction of HSC. However, much is still unknown about how the enzyme active site accommodates substrates of varying sizes during further oligomerization reactions. To answer this question, AMS analogs of the monomer (HSC-AMS), dimer (HSC-HSC-AMS), and trimer (HSC-HSC-HSC-AMS) were chemically synthesized. Biochemical results from in vitro DesD reactions, IC 50 assays, and isothermal titration calorimetry along with structural studies conducted via cocrystallization inform an updated mechanistic model for the iterative DesD catalytic cycle. The acyl adenylate motif in the growing substrate chain drives tight binding in the enzyme active site while the N-terminal HSC units dynamically sample conformations en route to terminating macrocyclization of the HSC-HSC-HSC trimer.