The first aryl acid-activating A domain structure was DhbE from the bacillibactin biosynthetic pathway, which selects for 2,3-dihydroxybenzoic acid (Dhb). The region that determines Dhb selectivity comprises residues H234-S240 (Figure ). Most if not all of the main chain atoms in DhbE seem to diverge from those of GrsA_A, despite the chemical similarities between Dhb and L-Phe. There is no conserved aspartate in DhbE because there is no α-amino group on the substrate to coordinate, which has previously caused some confusion due to the prevalence of D235 in most substrate selectivity codes. However, with the insight provided by structural data, namely the presence of a cisPro241 residue that shifts the peptide backbone, the corrected sequence alignments can now illuminate the importance of N235 and Y236 in Dhb binding (corresponding to D235 and A236 in GrsA_A). N235 forms a hydrogen bond with the 2'hydroxyl, while S240 hydrogen bonds with the 3'-hydroxyl moiety. Another core motif that confers aryl acid binding is A5, originally annotated as xNxYGPTExx in α-amino acid A domains, but strictly conserved as QQVxFMAEGL here. Randomizing the residues N235 and V337 in DhbE using yeast cell surface display and probe-based screening showed that the N235Q mutant displayed improved adenylation of 3-hydroxybenzoic acid, as did the A333S and A333T mutants. Interestingly, A333 was not previously thought to be part of the nonribosomal code for aryl acids. A333S and A333T also showed improved binding to 2-aminobutanoic acid (2-Aba), and selection with a 2-Aba probe further led to replacement of V337 with Lys or Arg. The Dhb-activating A domain EntE from the enterobactin pathway shows remarkable similarity to DhbE. Information gleaned from Dhb activation contributed to the engineering of EntE so that substrate binding pocket could be successful enlarged and allow for the incorporation of substituted benzoic acids. In particular, mutating the conserved D235, which binds to the 2hydroxyl moiety, to glycine (D235G) widened the entrance of the pocket. The EntE mutant was thus able to bind benzoic acids with bulky substituents at the 2 and 3 positions, namely 3cyanobenzoic acid and 2-nitrobenzoic acid, in the increased space this mutation provided. It was observed that the 2-nitro group specifically formed hydrogen bonding interactions with the backbone amides of Y236 and A335 (EntE numbering) .