Under iron deficient environment, strains of soil derived Streptomyces species have been reported to biosynthesize a mixture of desferrioxamines, a class of hydroxamate containing siderophores. The peptide backbone of desferrioxamine is assembled via iterative condensation of N-hydroxy-N-acyl-cadaverine by DesD, an ATP-dependent non-ribosomal-peptide-synthetase independent siderophore (NIS) synthetase. It is proposed that DesD catalyzes the activation of N-hydroxy-N-succinyl-cadaverine by forming an active acyl adenylate followed by condensation with the N⁵-amino group of a second molecule of the same precursor. Further iterations by DesD produce linear and cyclic trimeric desferrioxamine siderophores. In this work, we synthesized a structural mimic of the N-hydroxy-N-succinyl-cadaverine acyl adenylate in the form of an acyl sulfonyladenosine (AMS) analog. We obtained a high-resolution acyl-AMS bound crystal structure of DesD from Streptomyces griseoflavus providing good evidence for the formation of acyl adenylates during the DesD catalytic cycle. While acyl AMS derivatives can be potent inhibitors (nanomolar) against a variety of adenylate forming enzymes, the apparent IC50 values of the DesD acyl adenylate mimic was found to be ~50 micromolar against two enzyme orthologs. We attribute this low inhibitor potency to the iterative nature of DesD, which requires a high-degree of substrate plasticity to achieve consecutive enzymatic turnovers in a volume-controlled active site environment. Nonetheless, our AMS molecule serves as the first rationally designed inhibitor for a member of the NIS synthetase family of adenylating enzymes with utility as a chemical probe to provide unprecedented insight into the mechanism, function, and structure of this unique class of adenylating enzymes.

Gerry Rivera

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