Dissecting the Mechanism of Oligomerization and Macrocyclization Reactions of NRPS-Independent Siderophore Synthetases.

Macrocyclic and linear hydroxamate siderophores produced by NRPS-independent siderophore (NIS; NRPS=nonribosomal peptide synthetase) synthetases are important in the bacterial competition for iron, as virulence factors, and as drugs for medical use in humans. Despite their importance, the mechanistic details of NIS synthetases have so far remained obscure. Using synthetic substrate analogues as tools allowed for an interrogation of the mechanism of the two closely related NIS synthetases AvbD and DesD. While AvbD produces macrocyclic homo- and heterodimers as native products, DesD is responsible for the synthesis of trimeric desferrioxamines. These enzymes comprise two adjacent binding sites with different substrate selectivities, which direct oligomerization and macrocyclization steps. Exploiting this difference, synthetic substrates were used to invert the native affinities for the sites resulting in switching from trimerization to dimerization reactions for DesD. Based on this work, a comprehensive model explaining the mechanistic details of the reactions and the differences between trimerizing and dimerizing enzymes was developed. Finally, a DesD mutant demonstrated the tuneability of the enzyme's substrate selectivity by only minor changes in the protein sequence. This finding confirms the affinity-directed mechanism responsible for the iterativity of oligomerization and macrocyclization steps.

One Enzyme, Three Metabolites: Shewanella algae Controls Siderophore Production via the Cellular Substrate Pool.

Shewanella algae B516 produces avaroferrin, an asymmetric hydroxamate siderophore, which has been shown to inhibit swarming motility of Vibrio alginolyticus. We aimed to elucidate the biosynthesis of this siderophore and to investigate how S. algae coordinates the production of avaroferrin and its two symmetric counterparts. We reconstituted the reaction in vitro with the main enzyme AvbD and the putative biosynthetic precursors, and demonstrate that multispecificity of this enzyme results in the production of all three cyclic hydroxamate siderophores that were previously isolated as natural products from S. algae. Surprisingly, purified AvbD exhibited a clear preference for the larger cadaverine-derived substrate. In live cells, however, siderophore ratios are maximized toward avaroferrin production, and we demonstrate that these siderophore ratios are the result of a regulation on substrate pool level, which may allow rapid evolutionary adaptation to environmental changes. Our results thereby give insights into a unique evolutionary strategy toward metabolite diversity.