Adenylation domains of nonribosomal peptide synthases are presented with the task of activating and loading a single amino acid onto the free thiol of the phosphopantetheine cofactor of a thiolation domain, which ultimately results in the incorporation of one amino acid per peptide.  In the biosynthesis of certain nonribosomal peptides, it has been shown that some adenylation enzymes have an iterative function, resulting in the incorporation of two or more of the same amino acid into the final product (multiple functions). Alternatively, the colinearity rule suggests that, in some instances, an adenylation domain is able to activate and load different amino acids resulting in a peptide with amino acid substitutions at one position. In this case, the adenylation enzyme has relaxed substrate specificity but only incorporates one amino acid per peptide (single function). Here we demonstrate that NikE, an enzyme with sequence homology to adenylation domains of NRPS,  activates two unique organic acids in the biosynthesis of the dipeptide nikkomycin, and the activation of both of these organic acids is essential for the formation of the final product. Thus, NikE not only has dual substrate specificity but also a dual function. The results of these studies, along with other unusual features of NikE, will be presented, and the ramifications of these studies on how adenylation enzymes are characterized will be discussed.