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 (V_max = 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/PP_i-bound, and HSC-AMS/P_i-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 PP_i enhanced DesD inhibition by HSC-AMS, presumably via stabilization of the DesD–HSC-AMS complex, similar to the proposed mode of adenylate stabilization where PP_i 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.

铁载体是微生物用于环境铁螯合的有条件必需的代谢物。大多数链霉菌菌株产生基于异羟肟酸的去铁胺 (DFO) 铁载体，由N ¹ -羟基尸胺（或N ¹ -羟基腐胺）和琥珀酸的重复单元组成。DFO 生物合成操纵子desABCD在链霉菌中高度保守；然而，仅desABCD的表达并不能解释这种天然产物类别中的巨大结构多样性。在这里，我们报告了来自链霉菌的四种 DesD 直系同源物的体外重组和生化特征产生独特的 DFO 铁载体的菌株。在体外条件下，所有四种 DesD 直系同源物都显示出相似的饱和稳态动力学（V _max  = 0.9–2.5 μM·min ^-1）并产生大环三聚体 DFOE 作为首选产物，表明 DesD 在生物合成中的保守作用DFO 铁载体。我们进一步合成了N ¹ -羟基-N ¹ -琥珀酰尸胺 (HSC)-酰基-腺苷酸、HSC-酰基氨磺酰基腺苷类似物 (HSC-AMS) 的结构模拟物，并在 ATP 结合中获得了 DesD 的晶体结构、AMP/PP _i结合和 HSC-AMS/P _i结合形式。我们发现 HSC-AMS 抑制 DesD 直系同源物 (IC_{50 个}值 = 48–53 μM）导致线性三聚体 DFOG 和di -HSC 的积累，而牺牲了大环 DFOE。添加外源 PP _i增强了 HSC-AMS 对 DesD 的抑制作用，可能是通过稳定 DesD-HSC-AMS 复合物，类似于所提出的腺苷酸稳定模式，其中 PP _i仍然埋在活性位点中。总之，我们的数据表明，酰基-AMS 衍生物可用作化学探针和双底物抑制剂，以揭示这种独特的腺苷酸化酶家族的有价值的机制和结构洞察力。