biostudies-literatureAhmed SFU.S. Department of EnergyBasic Energy SciencesBiological and Environmental ResearchNational Institutes of HealthOffice of ScienceNational Institute of General Medical SciencesNIGMS NIH HHS108413https://www.ebi.ac.uk/biostudies/studies/S-EPMC12005286biostudies-literatureUnknown301(4)Siderophores are small molecule natural products that are often produced by enzymes called nonribosomal peptide synthetases that many pathogenic bacteria produce to adapt to low iron conditions. Nonribosomal peptide synthetase bioengineering could lead to the production of siderophore analogs with the potential to interrupt this unique bacterial iron uptake system, endowing the molecules with antimicrobial properties. Acinetobacter baumannii produces the catecholate siderophore acinetobactin to scavenge iron, a nutrient essential for several metabolic processes. Previous studies have reported synthetic analogs of acinetobactin that disrupt iron acquisition by A. baumannii, resulting in inhibition of bacterial growth. To foster a long-term goal of using a chemoenzymatic approach to produce additional analogs, we have targeted the adenylation domain BasE for the incorporation of alternate substrates. Here, we report a structure-guided approach to investigate the substrate selectivity of BasE for non-native aryl substrates. Using targeted mutagenesis in the active site of BasE, we generated mutants that catalyze the activation of alternate substrates with catalytic efficiencies comparable to the WT enzyme with its natural substrate 2,3-dihydroxybenzoic acid. We further solved structures of these mutants bound to the non-native substrates that illustrate an expanded binding pocket that support the improved promiscuity of BasE. Motivated to develop an approach to produce analogs of acinetobactin, including molecules that could block iron uptake or be readily conjugated to antibiotic cargo, our work aims to develop a structure-guided approach for using catecholate siderophore pathways to incorporate alternate substrates.The Journal of biological chemistryThe structural basis of substrate selectivity of the acinetobactin biosynthetic adenylation domain, BasE.PMC12005286P41 GM103393R35 GM136235GM-136235P41GM103393DE-AC02-76SF00515Ahmed SFGulick AMfalseThe structural basis of substrate selectivity of the acinetobactin biosynthetic adenylation domain, BasE.Siderophores are small molecule natural products that are often produced by enzymes called nonribosomal peptide synthetases that many pathogenic bacteria produce to adapt to low iron conditions. Nonribosomal peptide synthetase bioengineering could lead to the production of siderophore analogs with the potential to interrupt this unique bacterial iron uptake system, endowing the molecules with antimicrobial properties. Acinetobacter baumannii produces the catecholate siderophore acinetobactin to scavenge iron, a nutrient essential for several metabolic processes. Previous studies have reported synthetic analogs of acinetobactin that disrupt iron acquisition by A. baumannii, resulting in inhibition of bacterial growth. To foster a long-term goal of using a chemoenzymatic approach to produce additional analogs, we have targeted the adenylation domain BasE for the incorporation of alternate substrates. Here, we report a structure-guided approach to investigate the substrate selectivity of BasE for non-native aryl substrates. Using targeted mutagenesis in the active site of BasE, we generated mutants that catalyze the activation of alternate substrates with catalytic efficiencies comparable to the WT enzyme with its natural substrate 2,3-dihydroxybenzoic acid. We further solved structures of these mutants bound to the non-native substrates that illustrate an expanded binding pocket that support the improved promiscuity of BasE. Motivated to develop an approach to produce analogs of acinetobactin, including molecules that could block iron uptake or be readily conjugated to antibiotic cargo, our work aims to develop a structure-guided approach for using catecholate siderophore pathways to incorporate alternate substrates.2025-01-01T00:00:00Z2025 Apr2026-06-01T14:54:46.939Z2026-04-08T13:28:44.466ZS-EPMC120052864009688810.1016/j.jbc.2025.108413