Nanomolar inhibition of the enterobactin biosynthesis enzyme, EntE: Synthesis, substituent effects, and additivity

2,3-Dihydroxybenzohydroxamoyl adenylate (I) was prepared as a potential product analog inhibitor of EntE (EC# 2.7.7.58), a 2,3-dihydroxybenzoate AMP ligase from Escherichia coli that is required for the biosynthesis of enterobactin. This compound, obtained by the aqueous reaction of imidazole-activated adenosine 5′-phosphate and 2,3-dihydroxybenzohydroxamic acid, is a competitive inhibitor with a Ki value of 4.5 × 10−9 M. Deletion of the catecholic 3-OH group of (I), in compound (II), reduced inhibitory activity by a factor of 3.5, whereas, removal of both the 3-OH and 2-OH groups, in (III), reduced inhibitory activity by a factor of 2000. Acetohydroxamoyl adenylate (IV), in which the entire catechol moiety of (I) is replaced by a hydrogen atom, gave 10% inhibition at 6 × 10−4 M, indicating a reduction in affinity by more than 105. The binding free energy of (I) is nearly equivalent to the sum of the corresponding values for adenosine 5′-phosphate and 2,3-dihydroxybenzoate.