Dissecting the mechanism of oligomerization and macrocyclization reactions of NRPS independent siderophore synthetases

Abstract

Macrocyclic and linear hydroxamate siderophores produced by NRPS independent siderophore (NIS) 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 analogs as tools allowed 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. Concluding from our results, these enzymes comprise two adjacent binding sites with different substrate selectivities, which direct oligomerization and macrocyclization steps. Exploiting these difference, synthetic substrates were able to invert the native affinities for the sites resulting in switching from trimerization to dimerization reactions for DesD. Based on our results we developed a comprehensive model explaining the mechanistic details of the reactions and the differences between trimerizing and dimerizing enzymes. 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.