In salicylate (Sal) activating A domains, it has been proposed that S240 is replaced by a cysteine to limit the binding of 3-substituted aryl acids. Other residues proposed to mediate this change in selectivity are S239 and V330 (DhbE numbering). In a mutagenesis study, it was found that residue positions 236, 240 and 339 collectively regulated the specificity of A domains to either Dhb or Sal, with Dhb activation utilizing YSV and Sal activation requiring FCI across the three positions. The Sal-incorporating A domain CahJ from the cahuitamycin pathway was crystallized in the presence of different substituted monomers. Much like DhbE and EntE, CahJ maintains residue N236 to interact with the 2-hydroxyl, and as predicted, a C241 to sterically restrict the 3position, which is joined by L338 (Figure ). The residues surrounding the 4-, 5-and 6substituted positions around the Sal aromatic ring are C241/L338/V330, V330/G307, and G307/G308, respectively (CahJ numbering). Each of these interaction centers would allow for the binding of Sal analogs with methyl substituents at these positions, though slight rotations of the ring would be required for each, meaning that multiple neighboring methyl groups would not be tolerated. Indeed, the structures included in this report were bound to benzoate-AMP, Sal-AMP, 5-methyl-Sal-AMP, and 6-methyl-Sal-AMP.