Substrate specificity of PA1221. Specificity was tested utilizing the 32P-PPi exchange assay. Amino acids tested are reported as single letter code and background activity, lacking a carboxylate substrate, is labeled “0”. Additional acids were tested and labeled a, b, and c, representing acetate, 4-chlorobenzate, and 2-amino-benzoate, respectively. Three other assays were performed in a series of alternate experiments (Alt.). Two mutant enzymes were tested with valine as the amino acid substrate, the K499L A10 adenylation domain mutant (A) and the Ser533Ala phosphopantetheinyl attachment site mutant (S). Finally, the impact of 1.2-fold molar excess of the MbtH-like protein PA2412 is shown in the final bar (2).