An acyl-adenylate mimic reveals the structural basis for substrate recognition by the iterative siderophore synthetase DesD.

Siderophores are conditionally essential metabolites used by microbes for environmental iron sequestration. Most Streptomyces strains produce hydroxamate-based desferrioxamine (DFO) siderophores composed of repeating units of N1-hydroxy-cadaverine (or N1-hydroxy-putrescine) and succinate. The DFO biosynthetic operon, desABCD, is highly conserved in Streptomyces; however, expression of desABCD alone does not account for the vast structural diversity within this natural product class. Here, we report the in vitro reconstitution and biochemical characterization of four DesD orthologs from Streptomyces strains that produce unique DFO siderophores. Under in vitro conditions, all four DesD orthologs displayed similar saturation steady-state kinetics (Vmax = 0.9-2.5 µM⋅min-1) and produced the macrocyclic trimer DFOE as the favored product, suggesting a conserved role for DesD in the biosynthesis of DFO siderophores. We further synthesized a structural mimic of N1-hydroxy-N1-succinyl-cadaverine (HSC)-acyl-adenylate, the HSC-acyl sulfamoyl adenosine analog (HSC-AMS), and obtained crystal structures of DesD in the ATP-bound, AMP/PPi-bound, and HSC-AMS/Pi-bound forms. We found HSC-AMS inhibited DesD orthologs (IC50 values = 48-53 µM) leading to accumulation of linear trimeric DFOG and di-HSC at the expense of macrocyclic DFOE. Addition of exogenous PPi enhanced DesD inhibition by HSC-AMS, presumably via stabilization of the DesD-HSC-AMS complex, similar to the proposed mode of adenylate stabilization where PPi remains buried in the active site. In conclusion, our data suggest that acyl-AMS derivatives may have utility as chemical probes and bisubstrate inhibitors to reveal valuable mechanistic and structural insight for this unique family of adenylating enzymes.

Siderophore Synthetase DesD Catalyzes N-to-C Condensation in Desferrioxamine Biosynthesis.

Desferrioxamine siderophores are assembled by the nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase enzyme DesD via ATP-dependent iterative condensation of three N1-hydroxy-N1-succinyl-cadaverine (HSC) units. Current knowledge of NIS enzymology and the desferrioxamine biosynthetic pathway does not account for the existence of most known members of this natural product family, which differ in substitution patterns of the N- and C-termini. The directionality of desferrioxamine biosynthetic assembly, N-to-C versus C-to-N, is a longstanding knowledge gap that is limiting further progress in understanding the origins of natural products in this structural family. Here, we establish the directionality of desferrioxamine biosynthesis using a chemoenzymatic approach with stable isotope incorporation and dimeric substrates. We propose a mechanism where DesD catalyzes the N-to-C condensation of HSC units to establish a unifying biosynthetic paradigm for desferrioxamine natural products in Streptomyces.

Synthetic Mimics of Native Siderophores Disrupt Iron Trafficking in Acinetobacter baumannii.

Many pathogenic bacteria biosynthesize and excrete small molecule metallophores, known as siderophores, that are used to extract ferric iron from host sources to satisfy nutritional need. Native siderophores are often structurally complex multidentate chelators that selectively form high-affinity octahedral ferric iron complexes with defined chirality recognizable by cognate protein receptors displayed on the bacterial cell surface. Simplified achiral analogues can serve as synthetically tractable siderophore mimics with potential utility as chemical probes and therapeutic agents to better understand and treat bacterial infections, respectively. Here, we demonstrate that synthetic spermidine-derived mixed ligand bis-catecholate monohydroxamate siderophores (compounds 1-3) are versatile structural and biomimetic analogues of two native siderophores, acinetobactin and fimsbactin, produced by Acinetobacter baumannii, a multidrug-resistant Gram-negative human pathogen. The metal-free and ferric iron complexes of the synthetic siderophores are growth-promoting agents of A. baumannii, while the Ga(III)-complexes are potent growth inhibitors of A. baumannii with MIC values <1 µM. The synthetic siderophores compete with native siderophores for uptake in A. baumannii and maintain comparable apparent binding affinities for ferric iron (KFe) and the siderophore-binding protein BauB (Kd). Our findings provide new insight to guide the structural fine-tuning of these compounds as siderophore-based therapeutics targeting pathogenic strains of A. baumannii.