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; 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.

Iron-meditated fungal starvation by lupine rhizosphere-associated and extremotolerantStreptomycessp. S29 desferrioxamine production

“…S29 was able to produce twelve new acyl DFO-Bs similar to those previously described. 26,27 Of these 12, five were short acyl chain derivatives (C 3 , C 4 , C 4 hydroxylated, C 5 , and C 6 ), five were monounsaturated derivatives (uC 6 , uC 7 , uC 10 , uC 11 , and uC 12 ), and two were derivatives containing glutaric acid at the C-terminus, similar to those seen by Rütschlin et al 40 Pertaining to the unsaturated derivatives, placement of the alkene at the position α to the carbonyl is based off previous studies also reporting DFOs containing unsaturated acyl chains. 27,41 Additionally, the position of the hydroxy in the C 4 derivative was based on previous work showing multiple amphiphilic peptide-based siderophores contain hydroxylation at the β-position.…”

Iron-meditated fungal starvation by lupine rhizosphere-associated and extremotolerant Streptomyces sp. S29 desferrioxamine production

“…S29 was able to produce twelve new acyl DFO-Bs similar to those previously described. 26,27 Of these 12, five were short acyl chain derivatives (C 3 , C 4 , C 4 hydroxylated, C 5 , and C 6 ), five were monounsaturated derivatives (uC 6 , uC 7 , uC 10 , uC 11 , and uC 12 ), and two were derivatives containing glutaric acid at the C-terminus, similar to those seen by Rütschlin et al 40 Pertaining to the unsaturated derivatives, placement of the alkene at the position a to the carbonyl is based off previous studies also reporting DFOs containing unsaturated acyl chains. 27,41 Additionally, the position of the hydroxyl in the C 4 derivative was based on previous work showing multiple amphiphilic peptide-based siderophores contain hydroxylation at the b-position.…”

Novel South African Rare Actinomycete Kribbella speibonae Strain SK5: A Prolific Producer of Hydroxamate Siderophores Including New Dehydroxylated Congeners

“…These two enzymes are proposed to catalyze the decarboxylation of lysine 17 to form cadaverine 18 followed by hydroxylation to generate N -hydroxy-cadaverine 19 ( Figure 3 B). The acyltransferase, SpbC, is likely to catalyze the N -acylation of 19 [ 9 , 25 ], which then undergoes ATP-dependent oligomerization and macrocylization catalyzed by SpbD and SpbE to produce the N -hydroxy-containing hydroxamate desferrioxamines 2 – 6 [ 28 ].…”